HPV epitopes targeted by T cells infiltrating cervical malignancies for use in vaccines

ABSTRACT

The present invention relates to novel CD4+ and CD8+ T cell epitopes that are specific for HPV-specific E6 and E7 oncoproteins, to peptides comprising these novel T cell epitopes, and to (vaccine) compositions comprising these peptides for use in methods for the prevention and/or treatment of HPV related diseases. Preferred epitopes are recognized by a T cell that infiltrates a cervical neoplastic lesion or by a T cell from a draining lymph node, and are presented by an HLA-DQ or HLA-DP molecule, or an HLA-B.

RELATED APPLICATIONS

This present invention is a continuation patent application that claimspriority to PCT patent application number PCT/NL2008/050320, filed onMay 27, 2008, European Patent Application No. 07109281.1, filed on May31, 2007, European Patent Office Application No. 07109287.8, filed onMay 31, 2007, which claims the benefit of U.S. Provisional PatentApplication No. 61/941,070, filed on May 31, 2007, the entirety of whichare herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the fields of medicine and immunology.In particular it relates to novel HPV epitopes that may be used in theprevention, therapy and/or diagnosis of HPV associated diseases.

BACKGROUND OF THE INVENTION

Cervical cancer is the second most common cancer worldwide (Bosch et al.2003). High risk human papilloma virus (HPV) type 16 and 18 are thecause of cervical cancer in around two third of all patients (Bosch etal. 1995, Munoz et al. 2003). The HPV genome encodes two oncoproteins,E6 and E7, which are constitutively expressed in high grade cervicallesions and cancer because they are required for the onset andmaintenance of the malignant cellular phenotype (Zur Hausen, 1996).

The tumor-specific expression of these oncoproteins as well as thepresence of low levels of circulating E6- and E7-specific T cellsdetected in the peripheral blood of almost half of patients withcervical cancer (de Jong et al. 2004, van der Berg et al. 2001, Welterset al. 2003, Welters et al. 2006, Ressing et al. 1996, Bontkes et al.2000, Luxton et al. 1996) suggested that they could serve as tumorrejection antigens. However, the existence of circulating HPV-specific Tcells does not imply that they contribute to the anti-tumor response. Inorder to control the disease, these T cells should at least be able tohome to the tumor sites. Indeed, a proportion of cervical carcinomas areinfiltrated by lymphocytes (Bethwaite et al. 1996, Chao et al. 1999,Piersma et al. 2007) but in-depth knowledge on the specificity and typeof the T cells infiltrating these cervical tumors is still lacking,probably due to the relative difficulties to establish T cell culturesfrom tumor tissue. Nonetheless, a few early pioneers were able toisolate HPV-specific tumor infiltrating lymphocytes (TIL) from tumors,resulting in the identification of two single CD8⁺ T cell epitopes ofHPV16 (Evans et al. 1997, Oerke et al. 2005) and two CD4 T cell epitopesspecific for the less prevalent high risk subtypes HPV59 and HPV33 (Hohnet al. 1999, Hohn et al. 2000). However, larger studies on cervicaltissue-infiltrating lymphocytes are urgently needed to comprehend thecontribution and role of the HPV-specific adaptive immune response incervical cancer. In addition, this will allow the rational design ofsuccessful immune intervention strategies.

Recent studies showed that two cytokines, IL-7 and IL-15, have a majorrole in the expansion and survival of CD4⁺ and CD8⁺ effector memory Tcells. IL-7 provides survival signals for effector T cells (Li et al.2003). IL-15 is a critical growth factor in initiating T cell divisions,and in contrast to IL-2—which is generally used to expand TILcultures—does not limit continued T-cell expansion (Li et al. 2001).Furthermore, IL-15 can also act as an antigen-independent activator ofCD8(⁺) memory T cells (Liu et al. 2002). Together, IL-7 and IL-15 canexpand with very high efficiency effector memory T cells, while centralmemory T cells are less responsive and naive T cells fail to respond tostimulation with these cytokines (Geginat et al. 2001, McKinlay et al.2007, Bacchetta et al. 2002).

A number of previous studies have reported MHC class II restrictedrecognition of synthetic peptides consisting of sequences from in HPV 16E6 and/or E7 proteins by T cell from peripheral blood mononuclear cells(PBMC).

WO 02/070006 discloses a DR1 restricted response against a peptideconsisting of amino acids 127-142 of HPV16 E6 protein, a DQ2 restrictedresponse against a peptide consisting of amino acids 35-50 of HPV16 E7protein, a DR3 restricted response against a peptide consisting of aminoacids 43-77 of HPV16 E7 protein and a DR15 restricted response against apeptide consisting of amino acids 50-62 of HPV16 E7 protein.

Strang et al. disclose a DR7 restricted response in PBMC fromasymptomatic individuals against a synthetic peptide consisting of aminoacids 42-57 of HPV 16 E6 protein.

Altmann et al. discloses a response in PBMC from asymptomaticindividuals that are DR1/DR11-typed against a synthetic peptideconsisting of amino acids 5-18 of HPV16 E7 protein, a response in PBMCfrom asymptomatic individuals that are DR4/DR13-typed against asynthetic peptide consisting of amino acids 17-34 of HPV16 E7 proteinand a response in PBMC from asymptomatic individuals that areDR4/DR13-typed against a synthetic peptide consisting of amino acids69-82 of HPV16 E7 protein.

WO 02/090382 discloses the binding affinities for a series ofoverlapping peptides from HPV16 E6 and E7 proteins for HLA-DR moleculesthat are most prevalent in the caucasian population. WO 02/090382further reports responses against a number of the HPV16 E6 and E7peptides in CD8-depleted PBMC from patients with bowenoid papulosis.

There is however still a need for knowledge about the presence, type andspecificity of tumor infiltrating lymphocytes in HPV-associatedmalignancies, preferably for the more prevalent high risk subtypes suchas HPV16, 18, 31, 33 and 45. It is an object of the present invention toprovide for HPV epitopes that are targets for tumor infiltratinglymphocytes and that may be used in the prevention, therapy and/ordiagnosis of HPV associated diseases.

DESCRIPTION OF THE INVENTION

The present invention provides novel T cell epitopes that are identifiedon the basis of our analysis of the presence and HPV16 or HPV18specificity of cervix infiltrating T cells in a large group of 70patients with cervical malignancies. We found that these infiltratinglymphocytes comprise HPV-specific T cells. In more detailed analysis weidentified 17 novel CD4⁺ and CD8⁺ T cell epitopes and theirHLA-restriction elements but also revealed that HPV-specific immuneresponse directed towards all parts of the E6 and E7 oncoproteins.Unexpectedly, the vast majority of the CD4⁺ T cell epitopes werepresented in the context of the less abundantly expressed HLA-DQ andHLA-DP molecules. Since the identified T cell epitopes constitutephysiological targets in the immune response to HPV 16 and HPV 18positive tumors they are valuable targets for optimization of preventionagainst HPV-related diseases and immunotherapy in patients with HPVrelated diseases.

In one aspect, the present invention thus relates to amino acidsequences of newly identified CD4⁺ Th and CD8⁺ CTL cell epitopes of HPV,as well as HPV derived synthetic peptides and immunogenic compositionscomprising these are also part of the present invention. Such peptidesresult in a much improved, enhanced and prolonged CD8⁺ CTL effector andmemory response upon administration in a wide range of patients with HPVassociated disease, including HPV related malignancies. Such peptidescan also induce a much improved pro-inflammatory microenvironmnent thatis more likely to be infiltrated by effector cells, as the result ofthis CD4⁺ Th response.

Since the peptides of the invention are preferably used as a vaccinealone or in combination or as part of an immunogenic composition, thepeptides are preferably named vaccine peptides and the compositionvaccine compositions.

The use of relatively short peptides is highly preferred for medicalpurposes as these can be synthesized in vitro efficiently, which is notpossible or uneconomical for native proteins larger than about 100 aminoacids. Chemical synthesis of peptides is routine practice and varioussuitable methods are known to the skilled person. Chemical synthesis ofpeptides also overcomes the problems associated with recombinantproduction of intact proteins, which is difficult to standardize andrequires extensive purification and quality control measures. Peptideswith a length that exceeds the length of HLA class I and class IIepitopes (e.g. having a length as indicated below herein) areparticularly advantageous for use as vaccine component because they arelarge enough to be taken up by professional antigen presenting cells, inparticular DC, as explained in WO02/070006 and processed in the DCbefore cell surface presentation of the contained HLA class I and classII epitopes takes place. Therefore, the disadvantageous induction of Tcell tolerance by the systemic presentation of minimal HLA class Iepitopes on non-antigen presenting cells (as shown in Toes et al., 1996,Proc. Natl. Acad. Sci. U.S.A 93:7855 and Toes et al., 1996, J. Immunol.156:3911), is prevented by the application of peptides of the inventionhaving a length as indicated herein (as shown in Zwaveling et al., 2002,J. Immunol. 169:350). Peptides comprising epitopes which are to bepresented to T cell receptors of CTL and/or Th cells preferably havesufficient length to contain both HLA class I and HLA class II epitopes

In a first aspect of the invention there is provided a peptidecomprising a contiguous amino acid sequence selected from the fulllength amino acid sequences of at least one of the HPV E6 and E7proteins. Preferably, the contiguous amino acid sequence selected fromthe full length amino acid sequences of the HPV E6 and E7 proteins froma high risk HPV serotype, such as serotypes 16, 18, 31, 33 or 45, morepreferably from the amino acid sequences of the HPV E6 and E7 serotypes16, 18, 31 or 33, most preferably from serotypes 16 or 18, of which 16is most preferred. The amino acid sequence of the HPV serotype 16 E6 andE7 proteins are depicted in SEQ ID No. 1 and 2, respectively. The aminoacid sequence of the HPV serotype 18 E6 and E7 proteins are depicted inSEQ ID No. 3 and 4, respectively.

Preferably, the peptide comprises at least one HLA class II Th cellepitope and/or at least one HLA class I cytotoxic T cell epitope,preferably an epitope as herein defined below in more detail. Preferablythe peptide has a length of no more than 100 amino acids and comprisesat least 19 contiguous amino acids selected from the amino acid sequenceof one of the above-defined HPV proteins, wherein the peptide preferablycomprises at least one of an HLA class II epitope and an HLA class Iepitope, more preferably both at least one HLA class II epitope and atleast one HLA class I epitope and most preferably (but not necessarily)both from the amino acid sequence of one of the above-defined HPVproteins. More preferably, in the peptide at least one HLA class IIepitope and at least one HLA class I epitope are present within acontiguous amino sequence from the amino acid sequence of one of theabove-defined HPV proteins. For the sake of clarity, the peptides of theinvention preferably comprise HLA class I presented epitopes and/or HLAclass II presented epitopes. Each of these epitopes are presentable andwill bind to the corresponding specific HLA molecule present on thecells after having been processed as described herein. In the context ofthe invention, an HLA-haplotype specific epitope may therefore also bereferred to as an epitope binding to, presented by and/or beingrestricted by that HLA-haplotype.

Within the context of the invention, “a peptide has a length of no morethan 100 amino acids” preferably means that the number of consecutiveamino acids originating from a HPV protein and present in a peptide asdefined herein, is 100, 98, 96, 94, 92, 90 or less. Therefore, bydefinition, a peptide as defined herein is distinct from a full lengthHPV protein. Such a peptide may comprise additional amino acids than theones originating from a HPV protein or may entirely be made of orconsist of an amino acid sequence originating from a HPV protein. Thelength of the contiguous amino acid sequence from one of theabove-defined HPV proteins comprised within the peptide, preferably isat least 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acids and/orpreferably no more than 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89,88, 87, 86, 85, 84, 83, 82, 81, 80, 60, 50, 45, 40, 35, 33 or 30 aminoacids, more preferably the length of the contiguous amino acid sequencefrom one of the above-defined HPV proteins comprised within the peptideis 19-45, even more preferably 22-40 amino acids, even more preferably30-35 and most preferably 33-35 amino acids. In another preferredembodiment, the peptide of the invention consists of any of thecontiguous amino acid sequence from the HPV proteins as defined herein,whereby it is understood that no amino acids are appended to either endof the contiguous amino acid sequence from the HPV protein that are notcontiguous with this amino acid sequence in the sequence of the nativeHPV protein. The peptides of the invention may be easily synthesized andare large enough to be taken up by professional antigen presentingcells, processed by the proteasome and have sufficient physical capacityand length to contain at least one HLA class I and/or at least one HLAclass II epitope. Optionally a peptide may comprise N- or C-terminalextensions, which may be amino acids, modified amino acids or otherfunctional groups that may for instance enhance bio-availability,cellular uptake, processing and/or solubility.

A preferred peptide of the invention has a length of no more than 100,98, 96, 94, 92 amino acids and comprises at least 19 contiguous aminoacids from the amino acid sequence of at least one of an HPV E6 and E7protein, wherein the contiguous amino acid sequence comprises an epitopethat is recognized by a T cell that infiltrates a cervical neoplasticlesion or by a T cell that is present in or isolated from a lymph nodefrom the pelvic region, that is draining from the cervical neoplasticlesion, preferably a T cell that is present in or isolated from adraining lymph node comprising metastatic tumor cells. A peptideaccording to the invention is preferably used to induce a T-cellresponse.

In a further preferred peptide of the invention the contiguous aminoacid sequence comprises an epitope that is selected from the groupconsisting of amino acids 11-32 of an HPV E6 protein, amino acids 37-68of an HPV E6 protein, amino acids 52-61 of an HPV E6 protein, aminoacids 51-72 of an HPV6 protein, amino acids 55-86 of an HPV E6 protein,amino acids 61-82 of an HPV E6 protein, amino acids 71-92 of an HPV E6protein, amino acids 73-105 of an HPV E6 protein, amino acids 91-112 ofan HPV E6 protein, amino acids 101-122 of an HPV E6 protein, amino acids121-142 of an HPV E6 protein, amino acids 129-138 of an HPV E6 protein,amino acids 1-32 of an HPV E7 protein, amino acids 21-42 of an HPV E7protein, amino acids 51-72 of an HPV E7 protein, amino acids 76-86 of anHPV E7 protein; amino acids 13-22 of an HPV E6 protein, amino acids29-38 of an HPV E6 protein, amino acids 52-61 of an HPV E6 protein,amino acids 129-138 of an HPV E6 protein, amino acids 137-146 of an HPVE6 protein, amino acids 149-158 of an HPV E6 protein, and amino acids11-19 of an HPV E7 protein. In yet a further preferred peptide of theinvention the contiguous amino acid sequence comprises an epitope thatis selected from the group consisting of SEQ ID No.'s 5-26.

A preferred peptide of the invention comprises at least an HPV-specificclass II CD4⁺ Th cell epitope. Preferably, a class II CD4⁺ Th cellepitope comprised in a peptide according to the invention is capable ofinducing or activating a CD4⁺ Th cell in human patient with an HPVassociated disease and/or a healthy control. The activation ispreferably assessed ex vivo or in vivo, more preferably in a humanpatient with an HPV associated disease, such as an HPV associatedmalignancy, whose infected and/or tumor cells express an HPV protein asdefined above. Most preferably, the HLA class II epitope is capable ofactivating a CD4⁺ Th memory and/or CD4+ Th-effector response, i.e.activation of a CD45RO-positive CD4⁺ Th cell. This will lead, by virtueof the ‘license to kill’ signal through CD40-triggering of DC(Lanzavecchia, 1998) to a more robust CD8⁺ effector and memory CTLresponse. In another setting the activated CD4+ Th-cells may activatenon-HLA restricted killer cells of the immune system.

A preferred class II CD4⁺ Th cell epitope comprised in (a contiguoussequence in) a peptide according to the invention is selected from thegroup consisting of amino acids 11-32 of an HPV E6 protein, amino acids37-68 of an HPV E6 protein, amino acids 52-61 of an HPV E6 protein,amino acids 51-72 of an HPV E6 protein, amino acids 55-86 of an HPV E6protein, amino acids 61-82 of an HPV E6 protein, amino acids 71-92 of anHPV E6 protein, amino acids 73-105 of an HPV E6 protein, amino acids91-112 of an HPV E6 protein, amino acids 101-122 of an HPV E6 protein,amino acids 121-142 of an HPV E6 protein, amino acids 129-138 of an HPVE6 protein, amino acids 1-32 of an HPV E7 protein, amino acids 21-42 ofan HPV E7 protein, amino acids 51-72 of an HPV E7 protein, and aminoacids 76-86 of an HPV E7 protein. A more preferred class II CD4⁺ Th cellepitope comprised in (a contiguous sequence in) a peptide according tothe invention is selected from the group consisting SEQ ID No.'s 5-21.

Another preferred class II CD4⁺ Th cell epitope comprised in (acontiguous sequence in) a peptide according to the invention is anepitope that is restricted by a haplotype selected from the groupconsisting of DR4, DR7, DR12, DR15, DP1, DP0201, DP4, DP14, DP1401,DP17, DQ5, DQ6, DP1901, DQ*0301, DQ*0302, DQ*0308, DQ*0501. A furtherpreferred class II CD4⁺ Th cell epitope comprised in (a contiguoussequence in) a peptide according to the invention is an epitope that isrestricted by a DP or DQ haplotype, of which DP1, DP0201, DP4, DP14,DP1401, DP17, DQ5, DQ6, DP1901, DQ*0301, DQ*0302, DQ*0308, and DQ*0501are more preferred, One previously disclosed HLA-DQ restricted epitope(WO02/070006) consists of amino acid 35-50 of the HPV16 E7 protein. Thisepitope is however recognized epitope by peripheral T cells and not by aT cell that infiltrates a cervical neoplastic lesion or by a T cell thatis present in or isolated from a lymph node from the pelvic region, thatis draining from the cervical neoplastic lesion. The contiguous sequencein a peptide of the invention therefore preferably does not comprise anepitope consisting of amino acid 35-50 of the HPV16 E7 protein. Thus, apreferred class II CD4⁺ Th cell epitope comprised in (a contiguoussequence in) a peptide according to the invention is an epitope that isrestricted by a DP or DQ haplotype and not by a DR haplotype. Expressionof HLA-DR molecules is known to be upregulated on tumor cells.Presentation in that context may, as presentation of antigens onnon-professional Antigen Presenting Cells (APC), lead to induction oftolerance. Expression of HLA-DP or -DQ molecules is much lower butHLA-DQ and HLA-DP epitopes when presented on professional APC, such ase.g. DC, may nonetheless lead to effective immune responses.

Yet another preferred class II CD4⁺ Th cell epitope comprised in (acontiguous sequence in) a peptide according to the invention is anepitope that is restricted by a DP or DQ haplotype and that is anepitope of an HPV E6 or E7 protein, more preferably an E6 or E7 proteinof HPV serotypes 16, 18, 31, 33 or 45, and most preferably of HPVserotypes 16 or 18, of which 16 is most preferred.

Yet a further preferred class II CD4⁺ Th cell epitope comprised in (acontiguous sequence in) a peptide according to the invention is anepitope selected from the group consisting of amino acids 11-32 of anHPV E6 protein, amino acids 37-68 of an HPV E6 protein, amino acids52-61 of an HPV E6 protein, amino acids 51-72 of an HPV E6 protein,amino acids 61-82 of an HPV E6 protein, amino acids 71-92 of an HPV E6protein, amino acids 73-105 of an HPV E6 protein, amino acids 91-112 ofan HPV E6 protein, amino acids 101-122 of an HPV E6 protein, amino acids121-142 of an HPV E6 protein, amino acids 1-32 of an HPV E7 protein, andamino acids 51-72 of an HPV E7 protein. A more preferred class II CD4⁺Th cell epitope comprised in (a contiguous sequence in) a peptideaccording to the invention is selected from the group consisting SEQ IDNo.'s 5, 6, 7, 9, 10, 11, 12, 13, 16, 18, 19, 20 and 21.

In another preferred embodiment, a peptide of the invention comprises atleast an HPV-specific class I CD8⁺ CTL epitope. In addition, said HLAclass I epitope is preferably capable of activating a CD8⁺ CTL response.Most preferably, the CTL activating capability has been demonstrated exvivo and/or in vivo, in human healthy control individuals or even morepreferably in a human patient with an HPV associated disease, such as anHPV associated malignancy, whose infected and/or tumor cells express anHPV protein as defined above. The presence of both an HLA class I andclass II epitope within one peptide has been observed to be particularlyadvantageous due to synergy in mounting and maintaining an effective CTLcell response (as shown in Zwaveling et al., 2002).

Peptides comprising epitopes which are to be presented to T cellreceptors of CTL and/or Th cells preferably fulfill a number ofrequirements. The peptides preferably have sufficient length to containboth HLA class I and HLA class II epitopes. Furthermore, the peptidespreferably comprise anchor residues within their HLA class I bindingparts to enable binding to the class I molecules, respectively. Thestability of the interaction between peptide and presenting MHC moleculepreferably is sufficient in order to generate a significant andeffective immune response. In the context of the present invention, thestability of the interaction between peptide and presenting MHC moleculetherefore preferably is such that the peptide has an intermediate tohigh affinity binding, whereby an IC₅₀≦about 5 μM is considered highaffinity binding, about 5 μM<IC₅₀≦about 15 μM is considered intermediateaffinity binding, about 15 μM<IC₅₀≦100 μM is judged low affinity bindingand IC₅₀>about 100 μM was regarded as no binding, whereby the bindingaffinity of a peptide for an MHC molecule is determined as described invan der Burg et al., 1995 and Kessler et al., 2003.

A specific proteasomal cleavage site generating the C-terminus of theepitope, preferably is present exactly after the epitope amino acidsequence in order to be liberated from the larger peptide and presentedon the HLA class I molecule. Length requirements are much less strictfor HLA class II presented epitopes, therefore a need for preciseenzymatic generation of the class II binding peptide is less absolute.These requirements have been used in the present invention to localizeand design peptides in the full length sequences of HPV proteins,particularly in the HPV E6 and E7 proteins, which comprise preferred CTLand Th cell epitopes and/or combinations thereof and are thus highlysuitable peptides for vaccination purposes.

Moreover, in vitro and ex vivo T cell experiments are preferably used toconfirm the capability of peptides according to the invention to inducesubstantial CD4⁺ Th and CD8⁺ CTL responses. The peptides of the presentinvention thereby provide a marked improvement in the selection ofrelatively short peptides that may be chemically synthesized, comprisingthe most potent and most widely applicable HLA class I and/or class IIpresented T cell epitopes derived from the HPV E6 and E7 tumor antigens.The peptides are particularly optimized with respect to theirproteasomal cleavage and preferably contain at least one of HLA class Iand class II epitopes and more preferably both HLA class I and class IIepitopes. The liberation of the C-termini of CTL epitopes containedwithin the peptides of the invention by the 20S proteasome provides HLAclass I binding fragments with CD8⁺ CTL stimulatory capacity.

The HLA class I epitopes in the HPV peptides of the invention arepreferably capable of being presented on HLA alleles that arepredominant in the population of human subjects to be treated. PreferredHLA class I epitopes in HPV derived peptides of the invention areepitopes capable of binding to HLA-A2, HLA-B7, HLA-B14, HLA-B27,HLA-B57, and HLA*0201. The most preferred HLA class I CTL epitopes arethe HLA-B binding HPV epitopes, of which HLA-B7, HLA-B14, HLA-B27,HLA-B57 are most preferred. The HLA class I epitope preferably has ahigh peptide binding capacity (IC₅₀<about 5 μM peptide) or at leastintermediate affinity (5 μM<IC₅₀<about 15 μM peptide). A preferred classI CTL epitope comprised in (a contiguous sequence in) a peptideaccording to the invention is an epitope that is restricted by class Ihaplotype as indicated above and that is an epitope of an HPV E6 or E7protein, more preferably an E6 or E7 protein of HPV serotypes 16, 18,31, 33 or 45, and most preferably of HPV serotypes 16 or 18, of which 16is most preferred.

A preferred class I CTL epitope comprised in (a contiguous sequence in)a peptide according to the invention is selected from the groupconsisting of amino acids 13-22 of an HPV E6 protein, amino acids 29-38of an HPV E6 protein, amino acids 52-61 of an HPV E6 protein, aminoacids 129-138 of an HPV E6 protein, amino acids 137-146 of an HPV E6protein, amino acids 149-158 of an HPV E6 protein and amino acids 11-19of an HPV E7 protein. A more preferred class II CD4⁺ Th cell epitopecomprised in (a contiguous sequence in) a peptide according to theinvention is selected from the group consisting SEQ ID No.'s 7, 14,22-26.

A preferred epitope comprised in a peptide according to the invention isan epitope that is presented by an HLA-B molecule. Preferably, the HLA-Bmolecule is an HLA-B7, HLA-B14, HLA-B27 or HLA-B57 molecule. Suchepitope is selected from the group consisting of SEQ ID No.'s 7, 22, 24,25 and 26.

Another preferred epitope comprised in a peptide according to theinvention is an epitope that is presented by an HLA-A molecule.Preferably the HLA-A molecule is an HLA-A2, or HLA*0201 molecule. Suchepitope is selected from the group consisting of SEQ ID No.'s 23 and 26.

According to a more preferred embodiment, peptides of the invention havea length of no more than 100, 98, 96, 94, 94, 92 amino acids andcomprise a contiguous amino acid sequence from an HPV protein selectedfrom the group consisting of amino acids 1-32 of an HPV E6 protein,amino acids 19-50 of an HPV E6 protein, amino acids 41-65 of an HPV E6protein, amino acids 55-80 of an HPV E6 protein, amino acids 71-95 of anHPV E6 protein, amino acids 85-109 of an HPV E6 protein, amino acids91-122 of an HPV E6 protein, amino acids 109-140 of an HPV E6 proteinE6, amino acids 127-158 of an HPV E6 protein, amino acids 1-35 of an HPVE7 protein, amino acids 22-56 of an HPV E7 protein, amino acids 43-77 ofan HPV E7 protein, and amino acids 64-98 of an HPV E7 protein. Morepreferably the peptides of the invention consist of a contiguous aminoacid sequence from an HPV protein selected from the group consisting ofamino acids 1-32 of an HPV E6 protein, amino acids 19-50 of an HPV E6protein, amino acids 41-65 of an HPV E6 protein, amino acids 55-80 of anHPV E6 protein, amino acids 71-95 of an HPV E6 protein, amino acids85-109 of an HPV E6 protein, amino acids 91-122 of an HPV E6 protein,amino acids 109-140 of an HPV E6 protein E6, amino acids 127-158 of anHPV E6 protein, amino acids 1-35 of an HPV E7 protein, amino acids 22-56of an HPV E7 protein, amino acids 43-77 of an HPV E7 protein, and aminoacids 64-98 of an HPV E7 protein. The contiguous amino acid sequencefrom the HPV E6 or E7 proteins are preferably of HPV serotypes 16, 18,31, 33 or 45, and most preferably of HPV serotypes 16 or 18, of which 16is most preferred.

It is clear to a skilled person that a peptide as defined herein willhave a desired and advantageous property linked to the presence of anepitope in said peptide (for example an epitope which is identified inthe invention as being presented by at least one of an HLA-DQ and HLA-DPmolecule and/or as being recognized by a T cell that infiltrates acervical neoplastic lesion or by a T cell from a draining lymph node) assoon as this epitope is present in said peptide. A peptide according tothe invention is preferably used to induce a T-cell response.

The skilled person will understand that even if this application doesnot identify each peptide that can be designed as comprising orconsisting of a desired epitope as identified herein, nevertheless theinvention encompasses any peptide as defined herein comprising orconsisting of an epitope as identified herein. In a preferredembodiment, a peptide is distinct from a HPV protein. In anotherpreferred embodiment, a peptide does not comprise or consist of aminoacid 35-50 of the HPV16 E7.

For example, one preferred epitope is SEQ ID NO:5 (aa 11-32 of HPV16E6). This paragraph is illustrative and may be applied for each epitopeas identified herein. Any peptide comprising SEQ ID NO:5 is encompassedby the present invention and may be used according to the presentinvention. In this preferred embodiment, a peptide is distinct from aHPV protein. Preferred amino acid length for a peptide of the inventionhas already been defined herein. When designing a peptide of theinvention, a peptide may start at the N-terminal site of a given epitopeas identified herein or end at the C-terminal site of a given epitope asidentified herein. Alternatively, a given epitope (for example SEQ IDNO:5) may be comprised within a peptide of the invention. Using SEQ IDNO:5 as example, if we design a peptide having a length of 45 aminoacids, such peptide may consist or comprise 11-56, 1-45, 2-46, 3-47,4-48, 5-49, 5-50 from HPV16 E6. A peptide of the invention may furthercomprise any other HPV epitope as defined herein or as already known tothe skilled person.

In this preferred embodiment (SEQ ID NO:5 as epitope), a peptide doesnot comprise or consist of amino acid 9-33 of the HPV16 E6 as disclosedin US2005/0142541. In this preferred embodiment, a peptide does notcomprise or consist of amino acid 1-37 of the HPV16 E6 as disclosed inEP 451 550. In this preferred embodiment, a peptide does not comprise orconsist of amino acid 8-37 of the HPV16 E6 as disclosed in U.S. Pat. No.5,629,161. In a preferred embodiment, a peptide comprising SEQ ID NO:5consists of or comprises 10-32, 1-32, 1-45, 11-56, 2-46, 3-47, 4-48,5-49, 5-50 the numbers indicating the starting and ending amino acidfrom HPV16 E6

In another preferred embodiment (SEQ ID NO:8 as epitope, aa 55-86 fromHPV16 E6), a peptide does not comprise or consist of a fragment of HPV16 E6 as disclosed on uniprot having the following accession numberQ919B2 (1-99, numbers indicating the starting and ending amino acid fromHPV16 E6) or Q80882 (1-84). For this embodiment also, a peptidecomprising SEQ ID NO:8 may start at the N-terminal site of this epitope,or end at the C-terminal site of this epitope, or this epitope may bepresent within the peptide. For example if we design a peptide having alength of 45 amino acids, such peptides may consist or comprise 55-100,41-86, 45-90. In a preferred embodiment, a peptide comprising SEQ IDNO:8 consists of or comprises 55-100, 41-86, 45-90, the numbersindicating the starting and ending amino acid in the HPV 16 E6 proteinamino acid sequence.

The HPV-derived peptides of the invention may be modified by deletion orsubstitution of one or more amino acids, by extension at the N- and/orC-terminus with additional amino acids or functional groups, which mayimprove bio-availability, targeting to T-cells, or comprise or releaseimmune modulating substances that provide adjuvant or (co)stimulatoryfunctions. The optional additional amino acids at the N- and/orC-terminus are preferably not present in the corresponding positions inthe native amino acid sequence of the HPV protein, more preferably theyare not from any of the HPV E6 or E7 amino acid sequences (e.g. SEQ IDNo.'s 1-4). The skilled person will appreciate that HPV amino acidsequences of the various HPV serotypes are expressly included in theinvention.

The HPV-derived peptides of the invention are obtainable by chemicalsynthesis and subsequent purification (e.g. see Example 1). TheHPV-derived peptides of the invention are preferably soluble inphysiologically acceptable watery solutions (e.g. PBS) comprising nomore than 35, 20, 10, 5 or 0% DMSO. In such a solution the peptides arepreferably soluble at a concentration of at least 0.5, 1, 2, 4, or 8 mgpeptide per ml. More preferably, a mixture of more than one differentHPV-derived peptides of the invention is soluble at a concentration ofat least 0.5, 1, 2, 4, or 8 mg peptide per ml in such solutions.

A preferred use of the peptides according to the invention is their useas a medicament, whereby more preferably the peptides are used as avaccine or an active component thereof. Each peptide may be either usedalone or preferably in combinations of at least 2, 3, 4, 5, 6, 7, 8, 9,10, 12, 13, 15 and up to 20 different peptides of the invention, in thetreatment and/or prevention of cancer, for the manufacture ofmedicaments, preferably vaccine for the treatment or prevention of anHPV associated disease. Such a medicament and/or anti-tumor vaccineaccording to the invention may be used to treat patients suffering fromor at risk of developing the following, non extensive list of cervicalintraepithelial neoplasia of the cervix (CIN), vulva (VIN), vagina(VaIN), anus (AIN), and penis (PIN), as well as cancer of the cervix,vulva, vagina, anus, penis, and head & neck.

In a further aspect, the current invention further relates tocompositions which may be useful for treatment and/or vaccination ofhuman subjects, comprising at least at least 2, 3, 4, 5, 6, 7, 8, 9, 10,12, 13, 15 and up to 20 different peptides of the invention as definedabove and optionally one or more pharmaceutically acceptable excipients,in particular adjuvants and immune modulators. Preferably, thecomposition is a pharmaceutical composition and/or intended for use as amedicament. The pharmaceutical composition is preferably intended forvaccination. The pharmaceutical composition are preferably used for thetreatment and/or prevention of cancer, for the manufacture ofmedicaments, preferably vaccine for the treatment or prevention of anHPV associated disease. A non-exhaustive list of an HPV associateddiseases has already been given herein.

Thus, in one aspect the invention relates to the use of a peptide forthe manufacture of a medicament for the prevention and/or treatment ofan HPV associated disease, wherein the peptide has a length of no morethan 100, 98, 96, 94, 92 amino acids and comprises at least 19contiguous amino acids from the amino acid sequence of at least one ofan HPV E6 and E7 protein, wherein the contiguous amino acid sequencecomprises an epitope that is presented by at least one of an HLA-DQ andHLA-DP molecule. Preferably, the epitope is not the epitope presented inthe context of HLA-DQ2 and consisting of amino acid 35-50 of the HPV16E7 protein. Alternatively or in combination with previous preferredembodiment in another preferred embodiment, the contiguous amino acidsequence comprises an epitope that is recognized by a T cell thatinfiltrates a cervical neoplastic lesion or by a T cell from a draininglymph node. The peptides, contiguous amino acid sequences and epitopesare preferably as defined herein above.

In another aspect the invention relates to the use of a peptide for themanufacture of a medicament for the prevention and/or treatment of anHPV related disease, wherein the peptide has a length of no more than100, 98, 96, 94, 92, amino acids and comprises at least 19 contiguousamino acids from the amino acid sequence of at least one of an HPV E6and E7 protein, wherein the contiguous amino acid sequence comprises anepitope that is recognized by a T cell that infiltrates a cervicalneoplastic lesion or by a T cell from a draining lymph node. Thepeptides, contiguous amino acid sequences and epitopes are preferably asdefined herein above.

Formulation of medicaments, ways of administration and the use ofpharmaceutically acceptable excipients are known and customary in theart and for instance described in Remington; The Science and Practice ofPharmacy, 21^(st) Edition 2005, University of Sciences in Philadelphia.Pharmaceutical compositions and medicaments of the invention arepreferably formulated to be suitable for intravenous or subcutaneous, orintramuscular administration, although other administration routes canbe envisaged, such as mucosal administration or intradermal and/orintracutaneous administration, e.g. by injection. Intradermaladministration is preferred herein. Advantages and/or preferredembodiments that are specifically associated with intradermaladministration are later on defined in a separate section entitled“intradermal administration”.

It is furthermore encompassed by the present invention that theadministration of at least one peptide and/or at least one compositionof the invention may be carried out as a single administration.Alternatively, the administration of at least one peptide and/or atleast one composition may be repeated if needed and/or distinct peptidesand/or compositions of the invention may be sequentially administered.

The pharmaceutically compositions (also referred to as medicaments)according to the invention may preferably comprise at least one immuneresponse stimulating compound or adjuvant. Advantageously thepharmaceutical composition according to the invention may additionallycomprise one or more synthetic adjuvants. These adjuvants may be admixedto the pharmaceutical composition according to the invention or may beadministered separately to the mammal or human to be treated.Particularly preferred are those adjuvants that are known to act via theToll-like receptors and/or via a RIG-1 (Retinoic acid-Inducible Gene-1)protein and/or via an endothelin receptor. Immune modifying compoundsthat are capable of activation of the innate immune system can beactivated particularly well via Toll like receptors (TLR's), includingTLR's 1-10. Compounds capable of activating TLR receptors andmodifications and derivatives thereof are well documented in the art.TLR1 may be activated by bacterial lipoproteins and acetylated formsthereof, TLR2 may in addition be activated by Gram positive bacterialglycolipids, LPS, LPA, LTA, fimbriae, outer membrane proteins, heatshock proteins from bacteria or from the host, and Mycobacteriallipoarabinomannans. TLR3 may be activated by dsRNA, in particular ofviral origin, or by the chemical compound poly(I:C). TLR4 may beactivated by Gram negative LPS, LTA, Heat shock proteins from the hostor from bacterial origin, viral coat or envelope proteins, taxol orderivatives thereof, hyaluronan containing oligosaccharides andfibronectins. TLR5 may be activated with bacterial flagellae orflagellin. TLR6 may be activated by mycobacterial lipoproteins and groupB Streptococcus heat labile soluble factor (GBS-F) or Staphylococcusmodulins. TLR7 may be activated by imidazoquinolines. TLR9 may beactivated by unmethylated CpG DNA or chromatin—IgG complexes. Inparticular TLR3, TLR7 and TLR9 play an important role in mediating aninnate immune response against viral infections, and compounds capableof activating these receptors are particularly preferred for use in themethods of treatment and in the compositions or medicaments according tothe invention. Particularly preferred adjuvants comprise, but are notlimited to, synthetically produced compounds comprising dsRNA,poly(I:C), unmethylated CpG DNA which trigger TLR3 and TLR9 receptors,IC31, a TLR 9 agonist, IMSAVAC, a TLR 4 agonist, Montanide ISA-51,Montanide ISA 720 (an adjuvant produced by Seppic 7, France). RIG-1protein is known to be activated by ds-RNA just like TLR3 (Immunity,(2005), 1:19-28). In another preferred embodiment, the syntheticadjuvant compounds are physically linked to the peptides of theinvention. Physical linkage of adjuvants and costimulatory compounds orfunctional groups, to the HLA class I and HLA class II epitopecomprising peptides provides an enhanced immune response by simultaneousstimulation of antigen presenting cells, in particular dendritic cells,that internalize, metabolize and display antigen. Another preferredimmune modifying compound is an inhibitor of an endothelin receptor suchas BQ-788 (Buckanovich R J et al. Nature Medicine (2008), 14:28-36,Ishikawa K, PNAS (1994) 91:4892). BQ-788 isN-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D-1-methoxycarbonyltryptophanyl-D-norleucine.However any derivative of BQ-788 or modified BQ-788 compound is alsoencompassed within the scope of this invention.

Furthermore, the use of antigen presenting cell (co)stimulatorymolecules, as set out in WO99/61065 and in WO03/084999, in combinationwith the peptides and compositions of the invention is preferred. Inparticular the use of 4-1-BB and/or CD40 ligands, agonistic antibodies,OX40 ligands or functional fragments and derivates thereof, as well assynthetic compounds with similar agonistic activity are preferablyadministered separately or combined with the peptides of the inventionto subjects to be treated in order to further stimulate the mounting ofan optimal immune response in the subject.

In addition a preferred embodiment comprises delivery of the peptides,with or without additional immune stimulants such as TLR ligands and/oranti CD40/anti-4-1 BB antibodies in a slow release vehicle such asmineral oil (e.g. Montanide ISA 51) or PLGA. Alternatively, the peptidesof the invention may be delivered by intradermally, e.g. by injection,with or without immune stimulants (adjuvants). Preferably forintradermal delivery the peptides of the invention are administered in acomposition consisting of the peptides and one or more immunologicallyinert pharmaceutically acceptable carriers, e.g. buffered aqueoussolutions at physiological ionic strength and/or osmolarity (such ase.g. PBS).

Intradermal Administration

In a preferred embodiment, a peptide or a composition comprising apeptide or a medicament used in the invention all as defined herein areformulated to be suitable for intradermal administration or application.Intradermal is known to the skilled person. In the context of theinvention, intradermal is synonymous with intracutaneous and is distinctfrom subcutaneous. A most superficial application of a substance isepicutaenous (on the skin), then would come an intradermal application(in or into the skin), then a subcutaneous application (in the tissuesjust under the skin), then an intramuscular application (into the bodyof the muscle). An intradermal application is usually given byinjection. An intradermal injection of a substance is usually done totest a possible reaction, allergy and/or cellular immunity to it. Asubcutaneous application is usually also given by injection: a needle isinjected in the tissues under the skin.

In another further preferred embodiment, the medicament used in theinvention does not comprise any adjuvant such as Montanide ISA-51, itmeans the formulation of the medicament is more simple: an oil-waterbased emulsion is preferably not present in the medicament used.Accordingly, the medicament used in the invention does not comprise anadjuvant such as Montanide ISA-51 and/or does not comprise anoil-in-water based emulsion. Therefore, in a preferred embodiment, themedicament used in the invention is a buffered aqueous solutions atphysiological ionic strength and/or osmolarity, such as e.g. PBS(Phosphate Buffer Saline) comprising or consisting of one or morepeptide as defined earlier herein. The skilled person knows how toprepare such a solution.

The medicament as used in the invention has another advantage, which isthat by intradermally administering low amounts of a peptide as earlierherein defined, an immunogenic effect may still be achieved. The amountof each peptide used is preferably ranged between 1 and 1000 μg, morepreferably between 5 and 500 μg, even more preferably between 10 and 100μg.

In another preferred embodiment, the medicament comprises a peptide asearlier defined herein and at least one adjuvant, said adjuvant beingnot formulated in an oil-in water based emulsion and/or not being of anoil-in-water emulsion type as earlier defined herein. This type ofmedicament may be administered as a single administration.Alternatively, the administration of a peptide as earlier herein definedand/or an adjuvant may be repeated if needed and/or distinct peptidesand/or distinct adjuvants may be sequentially administered. It isfurther encompassed by the present invention that a peptide of theinvention is administered intradermally whereas an adjuvant as definedherein is sequentially administered. The adjuvant may be intradermallyadministered. However any other way of administration may be used forthe adjuvant.

The intradermal administration of a peptide is very attractive since theinjection of the vaccine is realized at or as close by as possible tothe site of the disease resulting in the local activation of the diseasedraining lymph node, resulting in a stronger local activation of theimmune system. In particular for VIN, VAIN, AIN, PIN, Penile cancer,Vulva cancer, Anal cancer, Head and Neck cancers.

In a preferred embodiment, the intradermal administration is carried outdirectly at the site of the lesion or disease. At the site of the lesionis herein understood to be within less than 5, 2, 1, 0.5, 0.2 or 0.1 cmfrom the site of the lesion.

Upon intradermally administering a medicament as defined herein, notonly Th2 but also Th1 responses are triggered. This is surprising sinceit was already found that cutaneous antigen priming via gene gun lead toa selective Th2 immune response (Alvarez D. et al, 2005 Furthermore, theimmune response observed is not only restricted to the skin as could beexpected based on (Alvarez D. et al, 2005). We demonstrate that specificT cells secreting IFNγ circulate through the secondary lymph system asthey are detected in the post challenged peripheral blood.

Another crucial advantage of the medicament of the invention is thatrelatively low amounts of peptides may be used, in one single shot, in asimple formulation and without any adjuvant known to give undesiredside-effects as Montanide ISA-51. Without wishing to be bound by anytheory, we believe the HPV intradermal peptide(s) used in the inventionspecifically and directly targets the epidermal Langerhans cells (LC)present in the epithelium. Langerhans cells are a specific subtype of DCwhich exhibit outstanding capacity to initiate primary immune responses(Romani N. et al 1992). These LC may be seen as natural adjuvantsrecruited by the medicament used in the invention.

In another preferred embodiment, the invention relates to the use of apeptide derived from HPV-E2, -E6 and/or -E7 protein for the manufactureof a medicament for the treatment or prevention of an HPV relateddisease, wherein the medicament is for intradermal administration asearlier defined and wherein in addition a peptide derived from HPV-E2,-E6 and/or -E7 protein is further used for the manufacture of amedicament for the treatment or prevention of an HPV related disease,wherein the medicament is for subcutaneous administration.

The medicament for intradermal administration has already been definedherein. The peptide used for subcutaneous adminstration is the same asthe one used for intradermal administration and has already been definedherein. The skilled person knows how to formulate a medicament suitedfor subcutaneous administration. Preferably, the medicament suited forsubcutaneous adminstration comprises a peptide as already herein definedin combination with an adjuvant. Preferred adjuvants have already beenmentioned herein. Other preferred adjuvants are of the type of an oil-inwater emulsions such as incomplete Freund's adjuvant or IFA, MontanideISA-51 or Montanide ISA 720 (Seppic France). In a further preferredembodiment, the medicament suited for subcutaneous administrationcomprises one or more peptides, an adjuvant both as earlier definedherein and an inert pharmaceutically acceptable carrier and/orexcipients all as earlier defined herein. Formulation of medicaments,and the use of pharmaceutically acceptable excipients are known andcustomary in the art and for instance described in Remington; TheScience and Practice of Pharmacy, 21^(nd) Edition 2005, University ofSciences in Philadelphia. The second medicament used in the invention isformulated to be suitable for subcutaenous administration.

In this preferred embodiment, the medicament suited for intradermaladministration may be simultaneously administered with the medicamentsuited for subcutaneous administration. Alternatively, both medicamentmay be sequentially intradermally and subsequently subcutaneouslyadministered or vice versa (first subcutaneous administration followedby intradermal administration). In this preferred embodiment as inearlier preferred embodiment dedicated to the intradermaladministration, the intradermal and/or subcutaneous administration of apeptide as earlier herein defined and/or of an adjuvant may be repeatedif needed and/or of distinct peptides and/or of distinct adjuvants maybe sequentially intradermally and/or subcutaneously administered. It isfurther encompassed by the present invention that a peptide of theinvention is administered intradermally and/or subcutaneously whereas anadjuvant as defined herein is sequentially administered. The adjuvantmay be intradermally and/or subcutaneously administered. However anyother way of administration may be used for the adjuvant.

We expect the combination of an intradermal and a subcutaneousadministration of a medicament according to the invention isadvantageous. DC in the epidermis are clearly different from DC in thedermis and in the subcutis. The intracutaneous (intradermal)immunization will cause antigen processing and activation of epidermalDC (Langerin-positive langerhans cells) that through their dendriticnetwork are in close contact with the keratinocytes. This will alsooptimally activate inflammatory pathways in the interactions betweenLangerhans cell and keratinocytes, followed by trafficking of antigenloaded and activated Langerhans cell to the skin-draining lymph nodes.

The subcutaneous administration will activate other DC subsets, thatwill also become loaded with antigen and travel independently to theskin-draining lymph nodes. Conceivably, the use of a medicament whichmay be administered both intradermally and subcutaneously may lead to asynergistic stimulation of T-cells in these draining nodes by thedifferent DC subsets.

In another aspect, the invention relates to nucleic acids encoding thepeptides and/or epitopes as defined herein above. Preferably the nucleicacids do not encode the wild type full length HPV E6 or E7 proteins butrather encode the peptides and/or epitopes of the invention as such, orflanked by amino acid sequence that are not contiguous with the wildtype HPV E6 or E7 proteins. Such flanking amino acids may be fromproteins other than the wild type HPV E6 or E7 proteins and/or they maybe from other locations within the wild type HPV E6 or E7 proteins thatare not contiguous with the peptide/epitope they flank. In a preferredembodiment the nucleic acids encode two or more peptides and/or epitopesof the invention arranged as beads-on-string, whereby the peptidesand/or epitopes of the invention (the beads) are linked directlytogether and/or are linked through linker sequences that are fromproteins other than the wild type HPV E6 or E7 proteins and/or fromother locations within the wild type HPV E6 or E7 proteins that are notcontiguous with the peptide/epitope they flank. The amino acid sequencesflanking or linking the peptides/epitopes may comprise proteolyticcleavage sites. Such nucleic acids may be applied to deliver thepeptides/epitopes of the invention in various ways. They may e.g. beused in the production of recombinant protein in a suitable host cell(e.g. E. coli) from which the may be purified. Alternatively the nucleicacid may be operably linked to expression regulatory sequences(promoters and the like) and incorporated in expression constructs forhuman cells. Such (autologous) cells may be transfected or transduced exvivo to be (re)-administered to a subject in need thereof. Alternativelythe expression construct may be incorporated into suitable gene therapyvector. Viral vector (based on a defective virus) are more efficientagents for gene transfer as compared to the non-viral agents. Suitableviral expression constructs include e.g. vectors that are based onadenovirus, adeno-associated virus (AAV), retroviruses or modifiedvaccinia Ankara (MVA).

In another embodiment, the present invention provides a tool to isolateHPV-specific T cell receptor (TCR) molecules from T cells capable ofinteracting with an HPV epitope of the invention as herein described. ATCR according to this invention will preferably be capable ofinteracting with the HPV epitope comprising peptides when they are inthe context of and/or displayed by an HLA molecule, preferably on aliving cell in vitro or in vivo. T cell receptors and in particularnucleic acids encoding TCR's according to the invention may for instancebe applied to transfer such a TCR into T cells from patients, whom areotherwise not capable to raise T cell immunity against an HPV epitopesof the invention as herein described. By this TCR cloning method, T cellclones may be provided that essentially are isogenic with the recipientto be treated with the T cell clones, i.e. the TCR expression T cellclones are autologous to the patient suffering from an HPV associateddisease. The method thus provides T cell clones capable of recognizingan HPV epitope according to the invention that may be generated for andcan be specifically targeted to tumor and/or HPV-infected cellsexpressing an HPV epitope in a subject in need thereof. In a preferredembodiment T-cells from the subject are isolated and transduced with theTCR recognizing the HPV epitopes of the invention as herein described.Following selection and expansion, known to the skilled artisan, theseautologous T cells that are now expressing a TCR which can recognizeHPV-induced tumor cells or HPV infected cells, can be re-infused intothe patient where they specifically target to the tumor and HPV infectedcells. Hence, the invention provides T lymphocytes encoding andexpressing a T cell receptor capable of interacting with an HPV epitopeas defined herein, preferably in the context of an HLA molecule. Said Tlymphocyte may be a recombinant or a naturally selected T lymphocyte. Tlymphocytes of the invention may also be used for or in the methods andpharmaceutical compositions of the invention. This specification thusprovides at least two methods for producing a cytotoxic T lymphocyte ofthe invention, comprising the step of bringing undifferentiatedlymphocytes into contact with an HPV epitope of the invention (or apeptide comprising the epitope) under conditions conducive of triggeringan immune response, which may be done in vitro or in vivo for instancein a patient receiving a graft, using peptides according to theinvention. Alternatively, it may be carried out in vitro by cloning agene encoding the TCR specific for interacting with an HPV epitope ofthe invention, which may be obtained from a cell obtained from theprevious method or which may be obtained from a subject exhibiting animmune response against the epitope, into a host cell and/or a hostlymphocyte, preferably a autologous lymphocyte, and optionallydifferentiate to cytotoxic T lymphocyte (CTL). Details of the methods inthis embodiment are described in e.g. De Witte et al. 2006 andSchumacher et al. 2002.

In a further embodiment the invention pertains to the use of the nucleicacids encoding the peptides and/or epitopes of the invention, T cellreceptors recognizing the epitopes of the invention, nucleic acidsencoding such T cell receptors, T cell (clones) expressing such nucleicacids as a medicament. Preferably the medicament is used in thetreatment and/or prevention of an HPV associated disease. Such amedicament according to the invention may be used to treat patientssuffering from or at risk of developing the following, non extensivelist of cervical intraepithelial neoplasia of the cervix (CIN), vulva(VIN), vagina (VaIN), anus (AIN), and penis (PIN), as well as cancer ofthe cervix, vulva, vagina, anus, penis, and head & neck.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition the verb “to consist” may be replaced by“to consist essentially of” meaning that a peptide or a composition asdefined herein may comprise additional component(s) than the onesspecifically identified, said additional component(s) not altering theunique characteristic of the invention. In addition, reference to anelement by the indefinite article “a” or “an” does not exclude thepossibility that more than one of the element is present, unless thecontext clearly requires that there be one and only one of the elements.The indefinite article “a” or “an” thus usually means “at least one”.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety. The followingexamples are offered for illustrative purposes only, and are notintended to limit the scope of the present invention in any way

DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B

FIG. 1A) Proliferation of initial T cell cultures isolated from cervicaltissue from 4 different patients. All T cell cultures recognizednaturally processed antigen in a 3-day proliferation assay uponstimulation with HPV16 or 18, E6 or E7 peptide pool and recombinantprotein. C265 recognized HPV16E6 peptide pool 1-92, C334 HPV16E6 peptidepool 71-158, C284 HPV16E7 peptide pool 1-98 and C228 HPV18E7 peptidepool 1-106. FIG. 1B) Fine mapping of the specificity of bulk culturesusing single peptides was measured by proliferation and IFNγ production.C265 responded to stimulation with peptide HPV16E6 37-68, C334 withHPV16E6 peptide 137-158, C284 with HPV16E7 peptide 71-92 and C228 withHPV18E7 peptide 21-42.

FIG. 2

Analysis of the type of T cell responding to HPV antigen as measured byintracellular cytokine staining for IFNγ. For positive peptide andprotein, the peptide HPV16E6 41-62 and HPV16E6 protein was used forC265, HPV16E6 protein and peptide 137-158 for C334, HPV16E7 protein andpeptide 71-92 for C284 and HPV18E7 protein and peptide 21-42 for C228.Peptides and proteins from HPV counterparts were used as negativecontrols. The TIL culture of C265 displayed a CD4⁺ and CD8⁺ T cellresponse which both responded to the HPV16 E6 41-62 peptide.

FIGS. 3A-3B

FIG. 3A) Blocking of CD4 restricted responses by HLA class II antibodiesin a 3-day proliferation assay. C265 derived T cells were stimulatedwith peptide loaded autologous B-LCL, C284 derived T cells werestimulated with peptide loaded monocytes that were matched only forHLA-DR12 and C228 derived T cells were stimulated with peptide loadedmonocytes, HLA-matched for DQ*0302. FIG. 3B) Finemapping and HLArestriction of TIL cultures. The CD4⁺ T cells of patient C265 werestimulated with autologous B-LCL pulsed with 10-mer peptides, coveringthe amino acid sequence of the recognized longer peptide, was tested inan ELISPOT assay. To determine the restriction of these CD4⁺ T cellsthey were stimulated with monocytes matched for HLA-DP2 only. FIG. 3C)Similarly, the minimal peptide-epitope recognized by the CD8 T cells ofC334 was determined by incubating these T cells with the indicated10-mer peptides in an ELISPOT assay. The HLA-restriction of C334 CD8⁺ Tcell response was determined using peptide pulsed PBMC isolated fromhealthy individuals whom were partially matched with the HLA class Imolecules of the patient.

FIGS. 4A-4D

Analysis of T cell reactivity present in tumor draining lymph node ofC427. FIG. 4A) Reactivity of T cell cultures after 3 weeks afterstimulation with HPV16E6 peptide pulsed autologous B-LCL measured in a3-day proliferation assay. FIG. 4B) Upper panel: recognition pattern ofthe T cell culture upon stimulation with autologous B-LCL pulsed withsingle 22-mer peptides. Lower panels: charting of the minimal epitoperecognized by T cell clones that were derived from this initial LNMCculture. CD4 T cell clone C427.47 was stimulated and tested in a 3 dayproliferation assay (left panel). The CD8 T cell clone C427.78 wastested in an IFNγ ELISPOT assay (right panel). FIG. 4C) The type of Tcell responding was determined by intracellular cytokine staining.HPV16E6 peptide 11-32 (upper panel) and peptide 137-158 (lower panel)were used as positive peptides. HPV18E7 peptide and protein were used asnegative controls. FIG. 4D) The restriction element was analyzed usingHLA class II blocking antibodies on partially matched B-LCL for class II(C427.47, upper panel) and on partially matched B-LCL for HLA class I(C427.78, lower panel), indicating that the CD4⁺ T cell response wasrestricted by HLA-DP14 and the CD8⁺ T cells by HLA-B14.

FIG. 5

An overview of the number, day of appearance and injected antigen thatinduced a positive skin reactions in the group of 19 healthy donors(HD). Skin reactions were considered positive when papules greater then2 mm in diameter arose no less then 2 days after injection. Theindicated layout is used for the 8 peptide pools, the first and lastamino acid in the protein of the peptide pool used is indicated. Thelayout printed in bold indicates at least one positive reaction withinthis timeframe; a filled square represents a new developed, positiveskin reaction to the indicated peptide pool.

FIG. 6

Detection of HPV16 specific T cells by IFNγ ELIspot in the pre-challengeblood sample of healthy donors is significantly correlated with theappearance of an early (<13 days) positive skin reaction to therecognized peptide pool (p=0.0003, two tailed Fisher's Extract test).Specific responses were calculated by subtracting the mean number ofspots+2×SD of the medium control from the mean number of spots inexperimental wells. The number of specific spots per 100.000 PBMC isgiven. Responses were considered positive if peptide pool specific Tcell frequencies were ≧5 in 100.000 PBMCs.

FIG. 7

A. Association between the appearance of a positive skin reaction andthe simultaneous detection (IFNγ ELlspot) of circulating HPV16 specificT cells in the post-challenge blood sample of healthy donors (p<0.0001,two tailed Fisher's exact test). From a total of 88 skin tests, 39 werepositive. Twenty-five of these 39 reactions were associated with apositive reaction in ELIspot (T cell frequency ≧5 in 100.000 PBMCs). Ofthe 49 skin test sites that did not show a skin reaction, 10 wereassociated with a positive ELIspot.

FIG. 8

A. HPV16 specific T cell responses detected by IFNγ ELIspot in thepost-challenge blood sample of healthy donors displaying a positive skinreaction. The mean number of spots per 100.000 PBMCs are depicted.Memory response mix (MRM) was used as a positive control. The filled barindicates the positive skin reaction site of which a punch biopsy wastaken and put in to culture.

B. T lymphocytes exfiltrating from punch biopsies were, after a 14- to28 day period of cytokine driven expansion, tested for their capacity toproliferate upon stimulation with monocytes pulsed with peptides (10μg/ml)—as injected in the skin test—or with protein (20 μg/ml).Phytohemagglutinine (PHA) served as a positive control. Proliferationwas measured by [³H]thymidine incorporation and a proliferative responsewas defined specific as the stimulation index (SI) ≧3. Healthy donor 17(HD17) is an example of a positive skin reaction site consisting of nonspecific T cells.

C. Supernatants of the proliferative responses in B were analysed forthe presence of IFNγ, interleukin 4 (IL4), IL5 and tumor necrosis factorα, IL2, IL10 (not shown) by cytometric bead array. Cutoff values werebased on the standard curves of the different cytokines (100 pg/ml IFNγand 20 pg/ml for the remaining cytokines). Antigen-specific cytokineproduction was defined as a cytokine concentration above cutoff leveland >2× the concentration of the medium control. Healthy donor 15 (HD15)displays a high background level of IL5, but is increased >2× afterantigen stimulation.

FIG. 9

T cell culture of the skin biopsy of pool 4 (E6₄₁₋₆₅, E6₅₅₋₈₀, E6₇₁₋₉₅)of healthy donor 15 (HD15) consists of both HPV16 specific CD4+ and CD8+T cells. The specificity of the culture was tested in an intracellularcytokine staining (ICS) against the protein (20 μg/ml) and the peptides(10 μg/ml) corresponding with the injected skin test. Remarkably, in 3out of 4 biopsies CD8+ HPV 16-specific T cells were detected.

EXAMPLES Example 1 Identification and Characterization of Novel HPVEpitopes

1. Methods

1.1 Subjects

Women presenting with histologically proven cervical neoplasia at thedepartment of Gynaecology of the Leiden University Medical Centre andLeyenburg Hospital the Hague were enrolled in the CIRCLE study, whichinvestigates cellular immunity against HPV16-positive cervical lesionsafter providing informed consent. The study design was approved by theMedical Ethical Committees of both hospitals. The subjects were testedfor HPV status using HPV 16 and HPV18 specific primers on DNA isolatedfrom surgical resection specimens (Claas et al. 1989). Peripheral bloodmononuclear cells (PBMC) for HLA-restriction analysis were obtained fromHLA-typed anonymous healthy blood donors after informed consent.

1.2 Antigens

A set of overlapping peptides spanning both HPV16 and HPV18 E6 and E7protein were used for T cell stimulation assays. HPV16 and HPV18 E6 andE7 consisted of 22-mers overlapping 12 residues. The peptides weresynthesized and dissolved as described earlier (van der Burg et al.2001, Welters et al. 2006). Recombinant HPV E6 and E7 proteins wereproduced in recombinant E. coli as described earlier (van der Burg etal. 2001). Moreover, a set of overlapping 10-mers (overlapping 9 aminoacids) of both HPV16 E6 and E7 was produced to pinpoint the minimalpeptide epitope recognized by HPV 16-specific T-cells.

1.3 Antigen Presenting Cells

Epstein-Barr virus transformed B cell lines (B-LCL) of the patients weremaintained in IMDM containing 10% FCS. Monocytes were generated fromperipheral blood lymphocytes as described earlier (de Jong et al. 2002).

1.4 Isolation and Culture of T Cells

Cervical tumor biopsies were obtained after radical hysterectomy,cervical neoplasia tissue was obtained from CIN III patients afterbiopsy. Fresh cervical tissue was minced in to pieces of approximately 1mm³ and cultured in IMDM (BioWhittaker, Verviers, Belgium), supplementedwith 10% human AB serum (Sigma, St. Louis Mo., USA), 10% T cell growthFactor (TCGF, Zeptometrix, Buffalo N.Y., USA) and 5 ng/ml IL-15(Peprotech, Rocky Hill N.J., USA). During the first day 5 ng/ml IL-7(Peprotech) was added to cultures to ensure T cell outgrowth. After 2-3weeks the specificity of the T cell (TIL, CIL) cultures was tested andpositive cultures were expanded using a mix of irradiated autologousB-LCL and 5 μg/ml cognate peptide.

Lymph nodes were derived from the pelvic region and contained tumorcells, indicative of metastatic cancer. The lymph nodes were cut intopieces and incubated for one hour at 37° C. in the presence ofcollagenase (200 IU/ml, Sigma) and DNAse (50 μg/ml, Sigma), after whichthe lymph node mononuclear cells were put through a cell strainer (BD,Erebodemgem, Belgium) to obtain a single cell suspension. Separate LMNCcultures were stimulated with HPV 16 or 18 E6 or E7 peptide pools andcultured for 2-3 weeks.

T cell clones were isolated using limiting dilution according to aprotocol adapted from Evans et al (Evans et al. 2001), replacing IL-2for 10% TCGF and 5 ng/ml IL-15, and adding 0.5 μg/ml phytohemagglutin(PHA, Murex Diagnostics, Dartford, UK) for T cell receptor triggering.After limiting dilution T cell clones were tested for their specificityand maintained in IMDM containing 10% Fetal Calf Serum (FCS, PAAlaboratories, Pasching, Austria), 10% TCGF and 5 ng/ml IL-15. T cellclones were expanded using a mix of culture medium, irradiated PBMC from3 different donors, B-LCL and 0.5 μg/ml PHA.

1.5 Analysis of T Cell Specificity

T cell cultures (25,000-50,000 cells/well) were tested on pulsedautologous monocytes or irradiated autologous EBVs for the recognitionof HPV 16 and 18 E6 and E7 peptides (5 μg/ml) and protein (10 μg/ml) intriplicate in a 3 day proliferation assay. After 48 hours supernatantwas harvested and stored at −20° C. for cytokine analysis. During thelast 16 hours of culture 0.5 μCi/well [³H]thymidine was added to measureproliferation (van der Burg et al. 2001). Antigenspecific IFNγproduction was measured by ELISA as described earlier (van der Burg etal. 1999).

MHC class II blocking experiments were performed as reported beforeusing murine monoclonal antibodies against HLA-DR (B8.11.2), HLA-DQ(SPV.L3) and HLA-DP (B7/21) (van der Burg et al. 1999). Peptide-pulsedAPC were incubated with anti-MHC class II antibodies for 2 hours priorto the addition of T cells.

Enumeration of IFNγ producing T cells as measured by intracellularcytokine staining was performed as described earlier (de Jong et al.2005). Briefly, APC were loaded with cognate peptide or recombinantprotein and incubated with T cell cultures. After 1 hour of incubation10 μg/ml Brefeldin A (Sigma) was added and incubated overnight.Hereafter the cells were fixed with 4% paraformaldehyde (Sigma) andpermeabilized with 0.1% Saponin. The samples were subsequently stainedwith CD4-APC, CD8-PerCP and IFNγ-PE and analyzed by flow cytometry. Theminimal peptide recognized by CD8 T cells was analysed by IFNγ ELISPOT(van der Burg et al. 2001, Welters et al. 2006, de Jong et al. 2002).CD8 T cell lines were seeded in triplicate wells at a density of 2×104on a Multiscreen 96-well plate (Millipore, Etten-Leur, The Netherlands)coated with an IFNγ catch antibody (Mabtech. Nacha, Sweden). Themicrocultures were stimulated with 5 μg/ml 10-mer peptides and incubatedovernight. Analysis of HLA restriction of CD8 T cells was performedusing 5 μg/ml 10-mer peptide pulsed PBMC or B-LCL co-cultured with equalnumbers of T cells. IFNγ specific spots were stained according to theinstructions of the manufacturer (Mabtech). The number of spots wasanalysed on a fully automated computer assisted video imaging system(BIOSYS).

2. Results

2.1 HPV-Specific T Cells are Present in Cervical Neoplasia InfiltratingLymphocytes

In the current study we analysed the presence, type and specificity ofHPV 16 and HPV 18-specific T cells in cervical neoplastic lesions, whichis the site where HPV-specific T cells encounter their cognate antigenand should exert their effector function. In total 74 patients wereanalyzed. Cervical tissue was obtained from 61 patients with cervicalcancer and from 9 additional patients with CIN III. Minced pieces oftissue were cultured for 2-3 weeks in the presence of a mix of cytokinescontaining IL-15 and TCGF. To prevent a potential bias in the outgrowthof tumor-specific T cells no exogenous HPV-antigens were provided tothese cultures. Within 14-21 days of culture the cytokine expanded Tcells were harvested and analysed by FACS. The mean percentage of CD3⁺ Tcells present in these cultures increased from 41% at 2 weeks to 68% at3 weeks. In general, the culture method did not favour the selectiveoutgrowth of one type of T cell as indicated by the percentage ofCD3+CD4+ T cells (34%±22%) and CD3⁺CD8⁺ T cells (52%±22%) at 2 weeks orat 3 weeks (38%±21%; 48%±24%, respectively). Occasionally, an individualculture showed a more pronounced expansion of either CD4⁺ or CD8⁺ Tcells (not shown). To analyze the presence of HPV-specific T cells, thecultures were stimulated with autologous monocytes pulsed with differentpools of overlapping peptides spanning the E6 and E7 proteins of HPV16and HPV18, as well as with the respective recombinant proteins. In 19 ofthe 51 HPV16- or HPV18-positive patients we were able to detectHPV-specific T cells by proliferation (Table 1, FIG. 1a ). Thesecultures responded both to peptide and protein loaded monocytes,indicating that the T cells recognized naturally processed antigen. In 8cultures E6-specific T cells were detected, in 10 cultures the T cellsresponded to E7 and in one T cell culture a response to both E6 and E7was detected. Importantly, no HPV16 or 18 specific T cell response wasdetected in HPV16 and 18 negative cervical tissues (n=19), indicatingthat the observed HPV16- and 18-specific responses were not induced invitro (Table 1).

2.2 Both HPV Specific CD4 and CD8 T Cells Infiltrate Tumor Tissue

Following the evaluation of HPV-specific reactivity, the 19 responding Tcell lines were expanded by stimulation with cognate peptide, cytokinemix and feeder cells. Fifteen of these HPV-specific cultures could besufficiently expanded for further analysis. The fine specificity of theHPV-specific T cells was determined in short-term stimulation assaysusing single peptides. Five cultures recognized 2 or more distinctpeptides, whereas the other 10 cultures recognized a single peptide(FIG. 1b , Table 1). To assess the type of T cell that responded toantigenic stimulation, the T cell cultures were stimulated with theircognate peptide and protein antigens and the response was analyzed byintra-cellular IFNγ staining (FIG. 2). The majority of the TIL culturescontained HPV-specific CD4+ infiltrating T lymphocytes (n=13 patients,13 different peptides recognized), whereas HPV-specific CD8⁺ T cellsinfiltrating lymphocytes were found in 6 cultures. In 9 of theHPV-specific T cell lines only a CD4⁺ T cell response was detected, in 4T cell lines both CD4⁺ T cells and CD8⁺ T cells reacted and in 2 T celllines only a CD8 T cells response was detected (Table 1, FIG. 2).

2.3 HLA Restriction of Tumor Infiltrating Lymphocytes

The HLA class I and II loci involved in the presentation of HPV peptidesto CD8⁺ T cells and CD4⁺ T cells were studied using blocking antibodiesand partially HLA matched APC isolated from healthy donors. A widevariety of HLA class II molecules were found to be involved in thepresentation of the antigens E6 and E7 of HPV16 and HPV18 (Table 2). Theuse of blocking antibodies against HLA-DR, HLA-DQ and HLA-DP revealedthat 3 of the detected responses were restricted by HLA-DR, 3 by HLA-DQand 3 by HLA-DP (FIG. 3a , Table 2). To determine the exact HLArestriction element involved in presentation of the HPV antigen, APCfrom healthy donors that are matched for only one HLA-allele were used(FIG. 3). In 6 cases we were not able to exactly determine therestriction element.

In case of patient C265 HPV-specific CD4+ and the CD8+ T cells bothresponded to the same peptide (FIG. 2). In order to discriminate betweenthese two T cell responses, T cell clones were established throughlimiting dilution. Unfortunately, only CD4⁺ T cell clones were obtainedand, as such, only the HLA class II-restriction element could beestablished. Therefore, it was only possible to determine the minimalpeptide and restriction in the other 5 different HPV-specific CD8 T cellcultures (Table 2). As an example, FIG. 3 shows the determination of theminimal peptide-epitope and restriction of the CD8 T cell response (FIG.3c ) of the TIL culture obtained from patient C334. This response wasrestricted by HLA-B27 as this CD8 T cell culture responded only uponstimulation with HLA-B27 matched peptide loaded APC and not with otherpartially HLA class I matched APC from other donors (FIG. 3c ). Onepatient (C265) displayed a CD8⁺ T cell response to two differentepitopes, and 2 patients (C176 and C334) responded to the sameHLA-B27-restricted CTL epitope (Table 2).

2.4 HPV-Specific T Cells in Tumor Draining Lymph Nodes

Tumor draining lymph nodes are the site where HPV-specific T cells areprimed and activated and, therefore, the HPV-specific T cell responsewas also studied in the tumor draining lymph nodes from 6 differentcervical cancer patients. Single cell suspensions of lymph nodemononuclear cells (LNMC) were isolated from cervical patients displayingmetastases in their lymph nodes. We were not able to directly detect HPVspecific responses ex vivo in freshly isolated LNMC (data not shown).Therefore, LMNC were first expanded by one round of in vitro stimulationwith HPV16 or 18 E6 and E7 peptide pools. In 4 cases the LNMC respondedto HPV16 and in 1 patient an HPV18 response was detected byproliferation and IFNγ production (Table 1, FIG. 4A). Similar to the TILcultures, patients with HPV16-positive tumors reacted only to HPV16whereas the patient diagnosed with an HPV18-positive cervical cancerreacted only against HPV18. No response to either HPV16 or HPV18 wasdetected in the LMNC from an HPV16/18-negative patient, despite the factthat the LNMC were stimulated with HPV 16 and HPV18 peptides in vitro(Table 1). T cell clones isolated from these LNMC cultures werecharacterized with respect to their fine specificity and HLA-restrictionelement. CD4⁺ T cell reactivities were found to 10 different peptides, 7of which were not detected in the TIL cultures. Three of these epitopeswere restricted by HLA-DQ and the other 4 by HLA-DP. In addition, oneHLA-A*0201-restricted and one HLA-B14-restricted CD8+ T cell epitope wasidentified (Table 2). FIG. 4 shows an example of the analysis of a LNMCculture. After one round of stimulation the LNMC cultures specificallyresponded to APC loaded with pools of HPV16E6 peptides or recombinantprotein (FIG. 4A). Analysis of the reactivity against single peptidesshowed recognition of a broad repertoire of peptides (FIG. 4B) and theCD4⁺ and CD8⁺ T cell clones isolated from this culture recognized theircognate antigen when naturally processed from recombinant protein (FIG.4 C). The restriction was further determined using HLA class II blockingantibodies and APC form partially matched donors (FIG. 4D).

Taken together, the analysis of both TIL and tumor-draining lymph nodecells revealed that in 23 of the 54 different HPV 16 or HPV 18 positivepatients a specific T cell response to in total 25 different E6- orE7-derived peptides can be detected. Notably, 13 CD4+ T cellpeptide-epitopes were restricted by HLA-DQ or HLA-DP, 3 by HLA-DR and in6 cases we were not able to distinguish between HLA-DQ/DP and HLA-DR(Table 2). Of the CD8⁺ T cell responses found, 2 were restricted byHLA-A, 4 by HLA-B and 2 were undetermined (Table 2).

3. Discussion

The HPV16 encoded oncoproteins E6 and E7 can serve as tumor rejectionantigens in animal models (Zwaveling et al. 2002, Peng et al. 2005)suggesting that they may also serve as target antigens fortumor-infiltrating lymphocytes in cervical cancer, but this has neverbeen systematically analyzed in a large group of patients. We were ableto establish a high number of TIL and CIN-infiltrating lymphocytes (CIL)cultures reactive against HPV16 and HPV18, which are the HPV types mostprominently associated with cervical cancer (Bosch et al. 1995, Munoz etal 2003). The cytokine mix used ensured the outgrowth of both CD4 andCD8 T cells without an overt preference for the expansion of either typeof T cell. In the course of our study 19 TIL cultures were establishedfrom patients diagnosed with a tumor positive for an HPV type other thanHPV16 or HPV18. None of these cultures reacted to stimulation with HPV16or HPV18 E6 and E7 antigens. Notably, TIL and CIL from HPV16-positivepatients did not respond to E6 and E7 of HPV18 and vice versa (Table 1).Therefore, the observed HPV-specific T cell responses in the TIL and CILof HPV16- or HPV18-positive patients are not the result of in vitroinduced T cell responses but a reflection of the anti-tumor response invivo. Recently, we showed that this protocol was also successful in theexpansion of TIL cultures from a small cohort of patients with ovariancancer (Lambeck et al. 2007).

Similar numbers of TIL cultures responded to E6 and E7 (Table 1).Identification of the cognate peptide-epitopes and HLA-restrictionelements of the HPV-specific immune responses revealed that HPV-specificimmunity was not restricted to a specific immunodominant region but wasaimed at all domains of the E6 and E7 oncoproteins (Table 2), suggestingthat both HPV E6- and E7-specific T cells will contribute to theanti-tumor response. Strikingly, our analysis revealed that the greatmajority of the HPV-specific CD4⁺ T cell responses were restricted byHLA-DQ or DP (13/16) and not by HLA-DR (Table 2). This was unexpectedbecause HLA-DR is the most abundant HLA class II molecule on the cellsurface of APC (Schwatz et al. 1988) as well as on cervical cancer cellswith de novo HLA class II expression (Hilders et al. 1994). Furthermore,in other tumor antigens most of the CD4⁺ T cell epitopes identified arepresented in the context of HLA-DR (80/93; see database onhttp://www.cancerimmunity.org). However, in cervical cancer there seemsto exist a more prominent role for HLA-DQ and HLA-DP restricted T cells,arguing that strategies, incorporating computer algorithms, to identifyfunctional T cell responses against HPV should not be focused on HLA-DRonly (Warrino et al. 2004, Facchinetti et al. 2005). In 7 patients aCD8⁺ T cell response was detected. In addition to the identification of3 novel HLA-B7, HLA-B14 and HLA-B27 restricted CD8 T cell epitopes, weconfirmed the presence of HLA-A*0201-restricted tumor-infiltrating CD8+T cells recognizing the HPV16 E7.11-20 epitope (Evans et al. 1997, Oerkeet al. 2005), albeit that stronger reactivity was observed against thepeptide sequence 11-19. In addition, CD8⁺ T cells reactive to theHLA-B57 restricted epitope HPV16E6.52-61 were detected. Based on thedetection of HLA-B57-restricted HPV16E6.52-61-specific CD8⁺ T cells inthe peripheral blood of healthy subjects it has been suggested that thisCTL epitope may play an important role in clearing HPV16-infection(Nakagawa et al 2004, Nakagawa et al 2007). However, the detection ofCTL responding to this epitope in cancer patients makes this lesslikely.

Our study shows that in at least 23 of the 54 different HPV16 or HPV 18positive patients, a specific T cell response to E6 and/or E7 can bedetected (Table 1). This will facilitate vaccination strategies aimingat the induction of a T cell response to these antigens to reinstate aneffective anti-tumor response in those patients with a pre-existingimmune response. Importantly, the T cell epitopes recognized by the Tcells in this study constitute physiological targets in the immuneresponse to HPV 16 and HPV 18 positive tumors. As such they will bevaluable for the integrated analysis of the magnitude and functionalityof HPV-specific T cell subsets at different stages of disease andmonitoring immunotherapy. The frequent presence of HPV-specific T cellsin cervical cancer patients may also constitute a valuable source oftumor-specific T cells that can be used in adoptive T cell transfertherapies.

Example 2 Intradermal Administration of a Peptide

Materials and Methods

Study Design

A cross-sectional pilot study to analyse HPV16 E2-, E6-, and E7-specificT-cell responses as measured by intradermal injection of pools ofclinical grade HPV 16 peptides in the upper arm was performed inpatients with HPV-related disorders of the cervix and in healthyindividuals. Since a delayed type hypersensitivity reaction represents amemory T-cell response, there was no prerequisite for HPV 16-positivityat the time of analysis.

Subjects

A group of nineteen healthy individuals (HD) participated in this studyafter providing informed consent. The group of healthy individualsdisplayed a median age of 31 years old (range, 20-51 years) and wascomprised of 80% women and 20% males. Peripheral blood mononuclear cells(PBMCs) were obtained from all subjects immediately beforeadministration of the skin test. The late appearance of positive skintests in healthy individuals resulted in the isolation of a second bloodsample from 11 of 19 healthy volunteers. The study design was approvedby the Medical Ethical Committee of the Leiden University MedicalCentre.

DTH Skin Test

Skin tests, based on Delayed Type Hypersensitivity reactions (DTH), canbe used as a sensitive and simple method for in vivo measurement ofHPV-specific cellular immune responses (Hopfl, 2000; Hopfl, 1991). Theskin test preparations consisted of 8 pools of long clinical-gradesynthetic peptides spanning the whole HPV 16 E6 and E7 protein and themost immunogenic regions of HPV 16 E2 protein (de Jong, 2004). Theseclinical grade peptides were produced in the interdivisionalGMP-Facility of the LUMC. Each pool of the skin test consisted of 2 or 3synthetic peptides, indicated by the first and last amino acid of theregion in the protein covered by the peptides. Pool 1: E2₃₁₋₆₀, E2₄₆₋₇₅,Pool 2: E2₃₀₁₋₃₃₀, E2₃₁₆₋₃₄₅, Pool 3: E6₁₋₃₁, E6₁₉₋₅₀, Pool 4: E6₄₁₋₆₅,E6₅₅₋₈₀, E6₇₁₋₉₅, Pool 5: E6₈₅₋₁₀₉, E6₉₁₋₁₂₂, Pool 6: E6₁₀₉₋₁₄₀,E6₁₂₇₋₁₅₈, Pool 7: E7₁₋₃₅, E7₂₂₋₅₆, Pool 8: E7₄₃₋₇₇, E7₆₄₋₉₈. Pool 3comprises Seq ID 5, 22 and 23. Pool 4 comprises Seq IDs 7-9. Pool 5comprises Seq IDs 11 and 12. Pool 6 comprises Seq IDs 13, 14, 24 and 25.Pool 7 comprises Seq ID 15 and 26. Pool 8 comprises Seq IDs 16 and 17.Per peptide pool 0.05 ml of 0.2 mg/ml peptides in 16% DMSO in 20 mMisotonic phosphate buffer (10 μg/peptide) was injected intracutaneously.The pools of peptides and a negative control (dissolvent only) wereinjected separately at individual skin test sites of the upper arm. Skintest sites were inspected at least three times, at 72 hours and 7 daysafter injection (Hopfl) of the peptides and at 3 weeks following thefirst report of a very late skin reaction in one of the first healthysubjects. Reactions were considered positive when papules greater than 2mm in diameter arose no less than 2 days after injection. From positiveskin reaction sites punch biopsies (4 mm) were obtained, cut in smallpieces and cultured in IMDM containing 10% human AB serum, 10% TCGF and5 ng/ml IL7 and IL15 to allow the emigration of lymphocytes out of theskin tissue. After 2 to 4 weeks of culture the expanded T cells wereharvested and tested for their HPV-specific reactivity.

Antigen for In Vitro Immune Assays

A set of peptides, similar to the peptides used in the skin test, wereused for T-cell stimulation assays and IFNγ-ELISPOT assays. The four HPV16 E2 peptides consisted of 30-mer peptides overlapping 15 residues, HPV16 E6 consisted of 32-mers and HPV 16 E7 of 35-mers, both overlapping 14residues. The peptides were synthesized and dissolved as previouslydescribed (van der Burg, 1999). Notably, in the IFNγ ELISPOT assayspeptide pool 4 and 5 slightly differed from the peptide pools used inthe skin test, pool 4 contained peptides E6₃₇₋₆₈, E6₅₅₋₈₆, E6₇₃₋₁₀₄ andpool 5 comprised peptides E6₇₃₋₁₀₄, E6₉₁₋₁₂₂.

Memory response mix (MRM 50×), consisting of a mixture of tetanus toxoid(0,75 Limus flocculentius/ml; National Institute of Public Health andEnvironment, Bilthoven, The Netherlands), Mycobacterium tuberculosissonicate (5 μg/ml; generously donated by Dr. P. Klatser, Royal TropicalInstitute, Amsterdam, The Netherlands), and Candida albicans (0.15mg/ml, HAL Allergenen Lab., Haarlem, The Netherlands) was used as apositive control. Recombinant HPV 16 E2, E6 and E7 proteins wereproduced in recombinant Escherichia coli as described previously (vander Burg, 2001).

Analysis of Antigen-Specific Th Cells by IFNγ ELISPOT

The presence of HPV 16-specific Th Cells was analyzed by ELISPOT asdescribed previously (van der Burg, 2001) Briefly, fresh PBMCs wereseeded at a density of 2×10⁶ cells/well of a 24-well plate (Costar,Cambridge, Mass.) in 1 ml of IMDM (Bio Whittaker, Verviers, Belgium)enriched with 10% human AB serum, in the presence or absence of theindicated HPV 16 E2, E6 and E7 peptide pools. Peptides were used at aconcentration of 5 μg/ml/peptide. After 4 days of incubation at 37° C.,PBMCs were harvested, washed, and seeded in four replicate wells at adensity of 10⁵ cells per well in 100 μl IMDM enriched with 10% FCS in aMultiscreen 96-well plate (Millipore, Etten-Leur, The Netherlands)coated with an IFNγ catching antibody (Mabtech AB, Nacha, Sweden).Further antibody incubations and development of the ELISPOT wasperformed according to the manufacturer's instructions (Mabtech). Spotswere counted with a fully automated computer-assisted-video-imaginganalysis system (Bio Sys). Specific spots were calculated by subtractingthe mean number of spots+2×SD of the medium control from the mean numberof spots in experimental wells (van der Burg, 2001).

T Cell Proliferation Assay

T-cell cultures of the skin biopsies were tested for recognition of thespecific peptides and protein in a 3-day proliferation assay (van derBurg, 2001). Briefly, autologous monocytes were isolated from PBMCs byadherence to a flat-bottom 96-well plate during 2 h in X-vivo 15 medium(Cambrex) at 37° C. The monocytes were used as APCs, loaded overnightwith 10 μg/ml peptide and 20 μg/ml protein. Skintest-infiltrating-lymfocytes were seeded at a density of 2-5×10⁴cells/well in IMDM suplemented with 10% AB serum. Medium alone was takenalong as a negative control, phytohemagglutinine (0.5 μg/ml) served as apositive control. Proliferation was measured by [³H]thymidine (5μCi/mmol) incorporation. A proliferative response was defined specificas the stimulation index (SI) ≧3. Supernatants of the proliferationassays were harvested 48 hours after incubation for the analysis ofantigen-specific cytokine production.

Analysis of Cytokines Associated with HPV16-Specific ProliferativeResponses

The simultaneous detection of six different Th1 and Th2 cytokines: IFNγ,tumor necrosis factor α, interleukin 2 (IL2), IL4, IL5 and IL10 wasperformed using the cytometric bead array (Becton Dickinson) accordingto the manufacturer's instructions. Cut-off values were based on thestandard curves of the different cytokines (100 pg/ml IFNγ and 20 pg/mlfor the remaining cytokines). Antigen-specific cytokine production wasdefined as a cytokine concentration above cutoff level and >2× theconcentration of the medium control (de Jong, 2004).

Intracellular Cytokine Staining (ICS)

The specificity and character of the T cell cultures derived frompositive skin reaction sites was tested by ICS as reported previously(de Jong, 2005). Briefly, skin test infiltrating lymphocytes wereharvested, washed and suspended in IMDM+10% AB serum and 2-5×10⁴ cellswere added to autologous monocytes that were pulsed overnight with 50 μlpeptide (10 μg/ml) or protein (20 μg/ml) in X vivo medium. Medium alonewas taken along as a negative control, phytohemagglutinine (0.5 μg/ml)served as a positive control. Samples were simultaneously stained withFITC-labelled mouse-antihuman IFNγ (0.5 g/ml, BD PharMingen),PE-labelled mouse-antihuman IL5 (0.2 mg/ml, BD PharMingen), APC-labelledanti-CD4 (BD Bioscience) and PerCP-labelled anti-CD8 (BD Bioscience).After incubation at 4° C., the cells were washed, fixed with 1%paraformaldehyde and analyzed by flow cytrometry (FACSscan, BDBiosciences)

Statistical Analysis

Fisher's Exact test (2-tailed) was used to analyze the relationshipbetween the detection of IFNγ-producing HPV-specific T-cells in PBMC,the presence of a skin test reaction or the presence of HPV-specificT-cells in skin biopsies, as well as differences between patients andhealthy controls with respect to the size or the number of the skinreactions within these groups. Statistical analyzes were performed usingGraphpad Instat Software (version 3.0) and Graphpad Prism 4.

Results

Skin Reactions to Intracutaneous Injection with HPV 16 E2, E6- and E7Peptides

We studied skin reactions in healthy subjects after intracutaneousinjection with HPV16 E2, -E6 and -E7 peptides. Positive skin reactionsappeared as flat reddish papules of 2 to 20 mm of diameter, arisingwithin 2 to 25 days after injection. A positive skin reaction wasdetected in 46 of the 152 skin tests in the healthy volunteers. Overall, each peptide-pool in the skin test could give rise to a positiveskin reaction. Most frequently reactions against E2₃₁₋₇₅ (10 out of 19subjects), E6₃₇₋₁₀₄ (9/16) and E7₄₃₋₉₈ (7/19) were observed in thecontrol group. This reaction pattern resembles that of what wepreviously observed in PBMC (de Jong, 2002; Welters, 2003) (FIG. 5).These skin reactions corresponded with the presence of a peptidespecific T cell response as detected in the PBMC of these individuals(data not shown).

Skin Reactions in Healthy Donors are Associated with Higher Frequenciesof HPV 16-Specific T-Cells in the Peripheral Blood.

In order to compare the results of the skin test with the presence ofcirculating HPV 16-specific type 1 T cells, an IFNγ ELIspot assay wasperformed with PBMC's collected before the intradermal peptide-challengewas given. In 5 out of 19 healthy volunteers we were able to detect aHPV 16-specific immune response by IFNγ-ELIspot. The detection of ≧5circulating HPV16-specific T-cells per 100.000 PBMC in the pre-challengeblood sample of healthy individuals was associated with an early (≦13days) positive skin reaction to the same peptide sequence (p=0.0003, twotailed Fisher's exact test; FIG. 6). No HPV16-specific circulatingT-cells were detected in the pre-challenge blood sample healthy donorsto peptides that induced a late positive skin reaction (14 to 25 days).This suggests that the frequency of circulating antigen-specific cellsdetermine the delay time for skin reactions to appear.

In order to assess the frequency of HPV-specific T-cells at the timethat a late skin reaction appeared additional blood samples from 11healthy volunteers were collected. In these individuals 39 out of 88skin tests were positive. In 25 of the 39 positive skin reactions and in10 of 49 negative skin reactions ≧5 HPV 16-specific T-cells weredetected per 100.000 PBMC. At this point a significant correlation wasfound between the detection of circulating HPV-specific IFNγ-producingT-cells in the post-challenged blood sample and the presence of a skinreaction (p<0.0001, Fisher's exact test; FIG. 7). This shows that thefrequency of HPV16-specific T cells in the blood of healthy volunteersis significantly higher following an intradermal challenge with HPV16peptide and indicates that intracutaneous injection of peptide antigensenhances the number of HPV16-specific T cells in the blood of healthyvolunteers.

Biopsies of Positive Skin Reaction Sites Consist of Both Th1/Th2-CD4+and CD8+ HPV16-Specific T Cells.

Approximately 25% of the positive skin reactions of healthy volunteerswere not associated with the detection of HPV16-specific IFNγ-producingT-cells in the blood, suggesting that other, non IFNγ-producing types ofT-cells may infiltrate the skin after intradermal injection of HPV16peptides.

In order to characterize the cells in a positive skin reaction sitepunch biopsies were taken. In total, 8 biopsies were taken fromdifferent positive skin reaction sites of 7 healthy controls andcultured with a cocktail of cytokines that allowed the outgrowth ofT-cells in vitro without antigenic stimulans. In 7 of 8 cases, T-cellsex-filtrated the tissue and expanded within 3-4 weeks. The expandedT-cells were tested for their specificity in a short term proliferationassay. FIG. 8 shows examples of T-cell cultures that specificallyproliferated upon stimulation with autologous monocytes pulsed with thepool of peptides, also injected in this site during the skin test (HD2,HD10, HD15) as well as to monocytes pulsed with HPV16 E6 protein (FIG.8AB). This indicates that these T-cells were capable of recognizingtheir cognate HLA-peptide complexes after the antigen was naturallyprocessed and presented. Analysis of the supernatants of theseproliferative T-cell cultures revealed a mixed Th1/Th2 cytokine profilein that the HPV 16-specific T-cells produced IFNγ, IL-4 and IL-5 (FIG.8C).

In each case that HPV-specific T-cells were detected in the biopsyculture (4 out of 8) this coincided with the detection of circulatingHPV16-specific IFNγ-producing T-cells in the post-challenge blood sampleby ELIspot (compare FIGS. 8A and B). In 3 of the other 4 positive skinreaction biopsies (HD2, HD17, HD18) the T-cells did not respond to HPV16peptides (FIG. 8; HD17) and in one case no T-cells ex-filtrated thetissue at all (HD13). In these 4 cases we were not able to detectcirculating HPV 16-specific IFNγ-producing T-cells in the post-challengeblood sample.

Co-staining of the biopsy-T cells by CD4 and CD8 cell surface markersshowed that not only HPV16-specific CD4⁺ but also HPV16-specific CD8⁺ Tcells infiltrated the skin site upon intradermal challenge with HPV 16peptide (FIG. 9). Overall, in 3 out of 4 biopsies infiltrated byHPV16-specific T-cells, we were able to detect HPV16-specific CD8⁺ Tcells. The CD8⁺ T cells isolated from the biopsy (pool 6) of HD2responded to both overlapping peptides of the injected skin test: HPV16E6₁₀₉₋₁₄₀ and E6₁₂₇₋₁₅₈ (data not shown), while the CD8⁺ T cells of bothsubjects HD15 and HD16 responded to HPV16 E6₃₇₋₆₈ (see example for HD15,FIG. 5).

Taken together, the population of immune cells migrating into the skinupon an intradermal challenge with HPV16 peptides comprisesHPV16-specific CD4⁺ Th1-, Th2- and CD8⁺ cytotoxic T cells. Thisinfiltration is paralleled by the appearance of circulatingHPV16-specific IFNγ-producing T-cells in the blood.

Discussion

Skin tests are commonly used as a simple assay for in vivo measurementof cell mediated immunity. We have validated the use of the skin testassay for the measurement of HPV 16 specific cellular immune responseagainst the early antigens E2, E6 and E7 in vivo by comparing theresults with that of parallel measurements of T cell reactivity by invitro assays.

In the group of healthy volunteers early skin reactions appeared between4 to 12 days after intradermal antigen challenge. In these individuals,known to display HPV 16 specific type 1 T cell responses in vitro (deJong, 2002; Welters, 2003), the appearance of an early skin reaction(within 13 days) was significantly associated with the detection ofIFNγ-producing HPV 16-specific T cells at a frequency of at least 1 per20.000 PBMC (FIG. 6, p<0.001). The same cut-off criteria for a positivereaction in the IFNγ ELIspot assay are recommended by Jeffries et al(Jeffries, 2006), who used mathematical tools to define the appropriatecut-off of the ELISPOT in relation to Mantoux-tests. The low number ofcirculating memory T cells (FIG. 6) may explain why the skin reactionsappear somewhat delayed compared to classical DTH tests. The T cellsneed to be boosted or reactivated and start to divide before enoughcells are produced to cause a local inflammatory reaction: the positiveskin test. Indeed, at the time a positive skin reaction appears, ahigher frequency of HPV 16-specific Th1 responses can be detected in theperipheral blood (FIG. 7).

Historically it has been postulated that the Th1 cell induce DTHresponses, however, several studies have now shown that also Th2 cellsinfiltrating the skin test sites (Wang, 1999; Woodfolk, 2001).Similarly, this study shows that the positive skin test sites of healthyvolunteers contain both Th1 and Th2 type HPV 16-specific T cells (FIGS.8 and 9). In addition, positive skin reactions may also be the result ofthe influx of non-specific T cells as became evident from two in depthstudies of positive skin test sites used to assay the specific immuneresponse following vaccination of patients with renal cell cancer ormelanoma (Bleumer, 2007). Also this study showed that a number ofpositive skin test sites from healthy subjects were infiltrated withT-cells that did not respond to the injected HPV16 antigens. So far, thereason for a-specific positive skin reactions remains unclear.Unexpectedly, we observed the majority of skin reactions in healthyindividuals to appear 2 to 3 weeks after intradermal injection of theantigen. While, these late positive skin reactions were not correlatedwith detection of circulating HPV-specific CD4⁺ memory T cells inpre-challenge blood (FIG. 6) the immunological constitution of theseskin test sites are similar to that of classic DTH tests (Platt, 1983;Poulter, 1982) and comprised of HPV16-specific CD4⁺ Th1- and Th2-cellsas well as HPV16-specific CD8⁺ T cells (FIGS. 8 and 9). We hypothesizethat these reactions might be the result of T cell priming. This hasalso been noted in 29% of patients whom underwent a 2-step tuberculinskin testing protocol and whom were only positive at the second testround (Akcay, 2003). In general, vaccine-induced T cell responses peakat 10 to 14 days after vaccination and not at three weeks. However, oneshould bear in mind that in such protocol a higher antigen dose as wellas strong adjuvants are injected. It is therefore reasonable to assumethat the T cell responses induced by intradermal challenge develop moreslowly and peak at a later period. Since the intradermal peptidechallenge in healthy volunteers results in the induction of both HPV16-specific CD4⁺ and CD8⁺ T cells it, therefore, should be considered asa single, low dose vaccination.

The main objective of this pilot study was to validate the use of theHPV16 specific skin test to detect type 1 immune responses in vivo. Inhealthy volunteers, a positive skin reaction within 13 days is indeedcorrelated with the presence of circulating IFNγ-producing memory Tcells as detected by the IFNγ ELIspot in vitro. Importantly, we alsoobserved discrepancies between the outcomes obtained by skin test andELIspot. In a number of cases HPV16-specific circulating IFNγ-producingT cells were detected in the post-challenge blood samples but without aconcomitant skin reaction and vice versa (FIG. 7), and this may beconsidered as a false negative or false positive result. In order tofully understand the impact of this on the interpretation of thedetection of type 1 immunity against HPV, we have begun a field trial ina large group of HPV positive patients and healthy volunteers inIndonesia.

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TABLE 1 HPV16 and 18-specific responses detected in infiltratinglymphocytes. No. HPV Stage of peptides Type of Status Origin Patient AgeCell Type disease Reactivity SI* recognized T cell HPV16 TIL 176 45squamous FIGO 1B E6 80 2 CD4/CD8 178 40 squamous FIGO 1B E7 11 1 CD4 18556 squamous FIGO 3B E7 6 1 CD8 192 37 squamous FIGO 1B 194 67 adeno FIGO2A E7 5 226 56 squamous FIGO 1B E6 3 1 CD4 229 42 squamous FIGO 1B 23045 squamous FIGO 1A 246 31 squamous FIGO 1B 265 44 squamous FIGO 1B E6104 2 CD4/CD8 267 49 squamous FIGO 1B E6 109 2 CD4 271 40 squamous FIGO1B 281 35 squamous FIGO 1B 283 51 squamous FIGO 1B 308 39 squamous FIGO1B 312 30 adeno FIGO 1B 331 65 squamous FIGO 1B E6 3 2 CD4/CD8 332 32squamous FIGO 1B 334 41 squamous FIGO 1B E6 5 1 CD8 338 34 squamous FIGO1B 340 29 squamous FIGO 1B 343 51 unknown FIGO 1B 344 43 squamous FIGO2A 363 45 squamous FIGO 1B 369 33 adeno FIGO 1A 371 31 squamous FIGO 1B372 72 squamous FIGO 1B 390 33 adeno FIGO 1B E6/E7 4 398 48 squamousFIGO 1B 405 41 squamous FIGO 2B 418 34 squamous FIGO 1B 415 46 squamousFIGO 1B 424 35 squamous FIGO 1B 441 51 squamous FIGO 1B 446 29 squamousFIGO 1B E6 4 4 CD4/CD8 CIL 279 60 unknown CIN3 284 36 squamous CIN2 E713 1 CD4 285 27 squamous CIN3 310 46 squamous CIN3 314 34 squamous CIN3E7 11 355 47 squamous CIN3 356 26 squamous CIN3 E7 3.5 1 CD4 LN 148 46squamous FIGO 1B E6/E7 9/3 CD4 267 49 squamous FIGO 1B E6 4 CD4 271 40squamous FIGO 1B E6/E7 1.5/2   CD4 427 28 squamous FIGO 1B E6 9 CD4/CD8HPV18 TIL 187 43 squamous FIGO 1B E6 2 1 CD4 196 48 adenosquamous FIGO2A 209 55 squamous FIGO 1B 214 42 adeno FIGO 1B E7 15 1 CD4 228 37squamous FIGO 2A E7 18 1 CD4 251 39 adenosquamous FIGO 2A E7 3 261 38squamous FIGO 1B 335 33 adeno FIGO 1B 378 40 adeno FIGO 1B E7 8 1 CD4 LN151 43 squamous FIGO 1B E6/E7 2/3 CD4 HPV16- TIL 181 40 squamous FIGO 1B18- 182 80 squamous FIGO 2B 215 31 squamous FIGO 1B 245 41 squamous FIGO1B 248 46 squamous FIGO 2A 264 35 adeno FIGO 1B 280 31 squamous FIGO 1B287 61 carcinosarcome FIGO 2B 289 45 adeno FIGO 1B 292 32 squamous FIGO1B 324 51 squamous FIGO 1B 353 35 adeno FIGO 1A 373 55 squamous FIGO 1B377 85 squamous FIGO 1B 381 80 adeno FIGO 1B 384 75 squamous FIGO 1B 41464 squamous FIGO 2A CIL 348 35 squamous CIN3 354 39 squamous CIN3 LN 42640 squamous FIGO 1B *SI = Stimulation Index of responding T cells

TABLE 2 T-cell epitopes recognized by cervical cancer patients T cellSEQ type epitope recognized restriction Origin patient ID CD4HPV16E6.11-32 DP17 LN C148  5 HPV16E6.11-32 DP1401 LN C271,  5 C427HPV16E6.37-68 DP0201 TIL C226  6 HPV16E6.52-61 DP0201 TIL C265  7HPV16E6.55-86 unknown LN, TIL C267  8 HPV16E6.61-82 DP1 or DP14 LN C427 9 HPV16E6.73-105 DP4 LN C148 10 HPV16E6.73-105 unknown LN, TIL C267 10HPV16E6.91-112 DR15 or DQ5 TIL C331 11 HPV16E6.91-112 unknown LN C267 11HPV16E6.101-122 DQ6 LN, TIL C427, 12 C446 HPV16E6.121-142 DP0201 or DQ5TIL C265 13 HPV16E6.121-142 unknown TIL C187 13 HPV16E6.129-138 DR7 TILC176 14 HPV16E7.21-42 DR4 TIL C178 15 HPV16E7.51-72 DP1901 CIL C356 16HPV16E7.76-86 DR12 CIL C284 17 HPV18E6.51-72 DQ*0301 LN C151 18HPV18E6.71-92 DQ*0501 LN C151 19 HPV18E7.1-32 DQ*0302, TIL C214 20DQ*0308 HPV18E7.1-32 unknown TIL C378 20 HPV18E7.21-42 DQ*0302 TIL C22821 CD8 HPV16E6.13-22 HLA-B7 TIL C446 22 HPV16E6.29-38 HLA-A2 LN C427 23HPV16E6.52-61 HLA-B57 TIL C331  7 HPV16E6.52-61 unknown TIL C265  7HPV16E6.129-138 unknown TIL C265 14 HPV16E6.137-146 HLA-B27 TIL C176, 24C334 HPV16E6.149-158 HLA-B14 LN C427 25 HPV16E7.11-19 HLA*0201 TIL C18526

What is claimed:
 1. A method for the treatment of an HPV-inducedintraepithelial neoplasia or cancer, comprising administering to a humansubject in need thereof an effective amount of at least two peptideshaving a length of no more than 45 amino acids, wherein each of the atleast two peptides comprises a different HPV E6 or E7 epitope selectedfrom the group consisting of SEQ ID Nos. 5, 6 9, 10, 11, 12, 13, 16, 18,19, 20 and
 21. 2. The method according to claim 1, and wherein theepitope is recognized by a T cell that infiltrates a cervical neoplasticlesion or by a T cell from a draining lymph node.
 3. The methodaccording to claim 1, wherein the epitope is of HPV E6 protein.
 4. Themethod according to claim 1, wherein the epitope is of HPV serotype 16,18, 31, 33 or
 45. 5. The method according to claim 4, wherein theepitope is of HPV serotype 16, 18, 31 or
 33. 6. The method according toclaim 5 wherein the epitope is of HPV serotype 16 or
 18. 7. The methodaccording to claim 1, wherein the peptides are of 22-35 amino acids inlength.
 8. The method according to claim 7, wherein the peptides are of33-35 amino acids in length.
 9. The method according to claim 1, whereinthe peptides are administered with at least one adjuvant.
 10. The methodaccording to claim 9, wherein the adjuvant acts via a Toll-likereceptor.
 11. The method according to claim 1, wherein the peptides areadministered intravenously, subcutaneously, intramuscularly, mucosally,intradermally and/or intracutaneously.
 12. The method according to claim1, wherein the peptides are administered with a pharmaceuticallyacceptable carrier.
 13. The method according to claim 1, wherein the atleast two peptides are selected from the group consisting of amino acids1-32 of HPV E6 protein comprising SEQ ID NO: 5; 91-122 of HPV E6 proteincomprising SEQ ID NO: 11; and 43-77 of HPV E7 protein comprising SEQ IDNO: 16; and combinations thereof.
 14. The method according to claim 13,wherein the protein is a protein of HPV serotype
 16. 15. The methodaccording to claim 1, wherein the HPV induced intraepithelial neoplasiacomprises intraepithelial neoplasia of cervix, vulva, vagina, anus, orpenis.
 16. The method according to claim 1, wherein the HPV-inducedcancer comprises cancer of cervix, vulva, vagina, anus, penis, head, orneck.
 17. The method according to claim 1, wherein the HPV E6 or E7epitopes are selected from the group consisting of SEQ ID Nos. 5, 11,12, and
 16. 18. The method according to claim 1, further comprising anadditional peptide having a length of no more than 45 amino acids andcomprising an epitope selected from the group consisting of SEQ ID Nos.5-26.